U.S. patent application number 12/805411 was filed with the patent office on 2011-12-15 for apparatus for single-molecule detection.
Invention is credited to Jung-Po Chen, Chung-Fan Chiou, Chang-Sheng Chu, Shuang-Chao Chung, Ming-Chia Li, Yu-Tang Li, Ying-Chih Pu, Chih-Tsung Shih, Rung-Ywan Tsai.
Application Number | 20110306143 12/805411 |
Document ID | / |
Family ID | 45096535 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306143 |
Kind Code |
A1 |
Chiou; Chung-Fan ; et
al. |
December 15, 2011 |
Apparatus for single-molecule detection
Abstract
An apparatus for detecting an object capable of emitting light.
The apparatus includes a light source and a waveguide. The
waveguide includes a core layer and a first cladding layer. At
least one nanowell is formed in at least the first cladding layer.
The apparatus further includes a light detector. The light detector
can detect a light emitted from a single molecule object contained
in the at least one nanowell.
Inventors: |
Chiou; Chung-Fan; (Hsinchu
City, TW) ; Tsai; Rung-Ywan; (Taoyuan, TW) ;
Li; Yu-Tang; (Tucheng City, TW) ; Shih;
Chih-Tsung; (Hsinchu City, TW) ; Li; Ming-Chia;
(Dajia Township, TW) ; Chu; Chang-Sheng; (Hsinchu
City, TW) ; Chung; Shuang-Chao; (Zhongli City,
TW) ; Chen; Jung-Po; (Toufen Township, TW) ;
Pu; Ying-Chih; (Tainan City, TW) |
Family ID: |
45096535 |
Appl. No.: |
12/805411 |
Filed: |
July 29, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12801503 |
Jun 11, 2010 |
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12805411 |
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Current U.S.
Class: |
436/94 ;
422/82.08; 422/82.11; 436/164; 506/39 |
Current CPC
Class: |
B82Y 15/00 20130101;
G01N 21/648 20130101; G01N 33/54373 20130101; G01N 21/7703
20130101; G01N 21/645 20130101; G01N 2021/6484 20130101; Y10S
977/958 20130101; Y10S 977/957 20130101; Y10S 977/954 20130101;
G01N 2201/088 20130101; G01N 2021/6482 20130101; Y10T 436/145555
20150115; G01N 21/6486 20130101; Y10T 436/143333 20150115 |
Class at
Publication: |
436/94 ;
422/82.11; 506/39; 422/82.08; 436/164 |
International
Class: |
G01N 33/00 20060101
G01N033/00; C40B 60/12 20060101 C40B060/12; G01N 21/64 20060101
G01N021/64; G01N 21/00 20060101 G01N021/00 |
Claims
1. A single molecule detection apparatus, comprising: a) a
waveguide comprising: a core layer; and a first cladding layer,
wherein at least one nanowell is formed in at least the first
cladding layer; and b) at least one light detector.
2. The detection apparatus of claim 1, further comprising a light
source.
3. The detection apparatus of claim 2, further comprising a light
coupler.
4. The detection apparatus of claim 1, wherein an upper opening of
the at least one nanowell is larger than a bottom of the at least
one nanowell.
5. The detection apparatus of claim 1, wherein an effective
excitation zone is formed near the bottom of the at least one
nanowell by a light field induced from a light wave propagating
along the core layer.
6. The detection apparatus of claim 1, wherein the at least one
nanowell and the detector are arranged at opposite sides of the
waveguide.
7. The detection apparatus of claim 1, wherein the light detector
generates a signal based on a detected light.
8. The detection apparatus of claim 1, wherein the waveguide is a
planar waveguide further comprising a second cladding layer, the
first cladding layer and the second cladding layer being arranged
at opposite sides of the core layer.
9. The detection apparatus of claim 1, wherein the at least one
nanowell extends through partial thickness of the first cladding
layer.
10. The detection apparatus of claim 1, wherein the at least one
nanowell extends through full thickness of the first cladding
layer.
11. The detection apparatus of claim 1, wherein the at least one
nanowell extends through full thickness of the first cladding layer
and partial thickness of the core layer.
12. The detection apparatus of claim 8, wherein the at least one
nanowell extends through full thickness of the first cladding layer
and full thickness of the core layer.
13. The detection apparatus of claim 1, wherein the light detector
comprises a set of optical sensors.
14. The detection apparatus of claim 1, further comprising a
plurality of nanowells forming a nanowell array.
15. The detection apparatus of claim 14, further comprising a
plurality of light detectors forming a detector array, each light
detector in the detector array corresponding to at least one
nanowell in the nanowell array.
16. The detection apparatus of claim 1, wherein a single molecule
object contained in the at least one nanowell absorbing an
excitation light further excites another single molecule object
through fluorescence resonance energy transfer so that the another
single molecule object emits light to be detected by the
detector.
17. The detection apparatus of claim 1, wherein the core layer has
a higher refractive index than the first cladding layer.
18. The detection apparatus of claim 8, wherein the core layer has
a higher refractive index than the first and second cladding
layers.
19. The detection apparatus of claim 1, wherein the core layer is
made from a material chosen from at least one of
Si.sub.xTi.sub.1-xO.sub.2, TiO.sub.2, Ta.sub.2O.sub.5,
Nb.sub.2O.sub.5, HfO.sub.2, Al.sub.2O.sub.3, ZrO.sub.2, ZnS,
SiN.sub.x, AlN.sub.x, TiN.sub.x, PC, PMMA, or Su8.
20. The detection apparatus of claim 1, wherein the first cladding
layer is made from a material chosen from at least one of
SiO.sub.2, MgF.sub.2, CaF.sub.2, Al.sub.2O.sub.3, PMMA, Su8, or
polycarbonate.
21. The detection apparatus of claim 8, wherein the first cladding
layer and the second cladding layer are made from a material chosen
from at least one of SiO.sub.2, MgF.sub.2, CaF.sub.2,
Al.sub.2O.sub.3, PMMA, Su8, or polycarbonate.
22. The detection apparatus of claim 1, further comprising a
protection layer arranged over the first cladding layer.
23. The detection apparatus of claim 22, wherein the protection
layer is opaque.
24. The detection apparatus of claim 23, wherein the protection
layer is made from a material chosen from at least one of Al, Ti,
Ni, Cr, Au, Ag, Cu, Pt, Pd, or an alloy of any two or more
thereof.
25. The detection apparatus of claim 8, further comprising an
opaque layer arranged between the second cladding layer and the
detector.
26. The detection apparatus of claim 25, wherein the opaque layer
is made from a material chosen from Al, Ti, Ni, Cr, Au, Ag, Cu, Pt,
Pd, or an alloy of any two or more thereof.
27. The detection apparatus of claim 25, wherein the opaque layer
has a hole arranged between the at least one nanowell and the light
detector so as to allow a light emitted from a single molecule
object contained in the at least one nanowell to reach the light
detector.
28. The detection apparatus of claim 25, wherein the opaque layer
has a nanopatterned structure arranged between the at least one
nanowell and the light detector so as to allow the transmission of
a light emitted from a single molecule object contained in the at
least one nanowell and block the transmission of noise.
29. The detection apparatus of claim 1, wherein an optical filter
is arranged between the waveguide and the light detector.
30. The detection apparatus of claim 8, wherein an optical filter
is arranged between the second cladding layer and the light
detector.
31. The detection apparatus of claim 25, wherein an optical filter
is arranged between the opaque layer and the light detector.
32. The detection apparatus of claim 8, wherein the second cladding
layer also serves as an optical filter.
33. The detection apparatus of claim 1, further comprising a cover
over the at least one nanowell.
34. The detection apparatus of claim 1, wherein at least a first
surface of the at least one nanowell has different surface property
than at least a second surface of the at least one nanowell.
35. The detection apparatus of claim 1, wherein a sidewall surface
of the at least one nanowell is hydrophilic, the sidewall surface
comprising a material chosen from silicon, silica, metal, or metal
oxide.
36. The detection apparatus of claim 1, wherein a bottom surface of
the at least one nanowell is hydrophobic.
37. The detection apparatus of claim 36, wherein the bottom surface
of the at least one nanowell is formed from a silicate or a metal
with a hydrophilic property modified, by R1.sub.x-Si(O--R2).sub.4-x
or a polymer with a functional group, to hydrophobic, and wherein
R1 is a hydrophobic group chosen from alkyl chain
--(CH.sub.2).sub.n--CH.sub.3 and R2 is C.sub.nH.sub.2n+1) and the
functional group is chosen from --COOH, --PO.sub.3H.sub.2, --SH, or
NH.sub.2.
38. The detection apparatus of claim 36, wherein the bottom surface
of the at least one nanowell is formed from a metal oxide with a
hydrophilic property modified, by R1.sub.x--Si(O--R2).sub.4-x or a
polymer with a functional group, to hydrophobic, and wherein R1 is
a hydrophobic group chosen from alkyl chain --(CH.sub.2).sub.n--CH3
and R2 is C.sub.nH.sub.2n+1, and the functional group is chosen
from --COOH, --PO.sub.3H.sub.2, --SH, or NH.sub.2.
39. The detection apparatus of claim 1, wherein a bottom of the at
least one nanowell has a different surface property from that of a
sidewall surface of the at least one nanowell.
40. The detection apparatus of claim 34, wherein the surface
property comprises hydrophobicity, functional group, functional
group density, material density, or conductivity.
41. The detection apparatus of claim 39, wherein the surface
property comprises hydrophobicity, functional group, functional
group density, material density, or conductivity.
42. The detection apparatus of claim 3, wherein the light coupler
is a grating coupler located over the core layer, in the core
layer, or beneath the core layer.
43. The detection apparatus of claim 3, wherein the light coupler
is a prism coupler.
44. The detection apparatus of claim 3, wherein the light coupler
is a side coupler.
45. The detection apparatus of claim 3, wherein the light coupler
is an absorbing-emitting coupler, which absorbs an incident light
emitted from the light source and emits an excitation light.
46. The detection apparatus of claim 45, wherein the
absorbing-emitting coupler comprises a photoluminescent material
having a Stokes shift greater than or equal to 30 nm.
47. The detection apparatus of claim 46, wherein the
photoluminescent material is doped in a polymer.
48. The detection apparatus of claim 46, wherein the
photoluminescent material comprises a photoluminescent dye, the
photoluminescent dye comprising anthracene, coumarin, pyrene,
stilbene, porphyrin, perylene, Alq3, eosin, Bodipy dye,
fluorescein, rhodamine, polymethine dye, DCM, or its
derivative.
49. The detection apparatus of claim 46, wherein the
photoluminescent material comprises a photoluminescent polymer, the
photoluminescent polymer comprising PPV or its derivative.
50. The detection apparatus of claim 46, wherein the
photoluminescent material comprises an inorganic compound, the
inorganic compound comprising quantum dot, alumina oxide, or zinc
oxide.
51. The detection apparatus of claim 3, wherein the light coupler
is a co-directional coupler.
52. The detection apparatus of claim 2, wherein the light source is
a laser, a laser diode, an LED, an OLED, a QLED, a fiber light
source, or an arc discharge fluorescent lamp.
53. The detection apparatus of claim 1, wherein the first cladding
layer has a refractive index approximately equal to that of a
sample solution.
54. The detection apparatus of claim 1, wherein the first cladding
layer has a refractive index larger than that of a sample
solution.
55. The detection apparatus of claim 1, wherein a size of a bottom
of the at least one nanowell is smaller than a wavelength of an
excitation light in the core layer, smaller than one-half of the
wavelength, smaller than one-quarter of the wavelength, or smaller
than one-eighth of the wavelength.
56. The detection apparatus of claim 2, wherein the light source is
formed on a supporting substrate of the waveguide.
57. The detection apparatus of claim 1, wherein the light detector
is an optical sensor chosen from a CCD, a CMOS optical sensor, a
photoconductive type optical sensor, a photovoltaic type optical
sensor, an APD, a p-n photodiode, a p-i-n photodiode, or a
multi-junction photodiode.
58. The detection apparatus of claim 1, wherein the light detector
is a set of optical sensors chosen from CCDs, CMOS optical sensors,
photoconductive type optical sensors, photovoltaic type optical
sensors, APDs, p-n photodiodes, p-i-n photodiodes, or
multi-junction photodiodes.
59. The detection apparatus of claim 58, wherein the optical
sensors in one set have different sensitive wavelength band.
60. A method of detecting a single molecule, comprising: emitting,
by a light source, an incident light; coupling, by a coupler, the
incident light into a waveguide, forming an excitation light in the
waveguide; forming, by the excitation light and at least one
nanowell formed in at least a cladding layer of the waveguide, an
effective excitation zone; and exciting, by the excitation light, a
single molecule object in the effective excitation zone, to cause
the single molecule object to emit a light to be detected by a
light detector.
61. A method of sequencing a nucleic acid, comprising the steps of:
providing a detection apparatus comprising: a waveguide comprising:
a core layer; and a first cladding layer; wherein at least one
nanowell is formed in at least the first cladding layer; and at
least one light detector; providing at least one nucleic acid
molecule; locating the at least one nucleic acid molecule
individually within the at least one nanowell; performing single
molecule sequencing-by-synthesis of the at least one nucleic acid
molecule, wherein the single molecule nucleic acid
sequencing-by-synthesis leads to emission of light correlated to
the identity of at least one base in the nucleic acid; detecting
the light with the detector, resulting in an output signal; and
processing the output signal to determine an identity of at least
one base comprised by the nucleic acid.
62. The method of claim 61, wherein the single molecule nucleic
acid sequencing-by-synthesis leads to emission of light via
chemiluminescence.
63. The method of claim 61, wherein the detection apparatus further
comprises a light source and the single molecule nucleic acid
sequencing-by-synthesis leads to emission of light via
fluorescence.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to a detection apparatus, and
the method of using the apparatus to detect an object. Further, the
present disclosure relates to a detection apparatus that is able to
detect a light of low intensity emitted from an object, such as a
single molecule object.
BACKGROUND
[0002] The Human Genome Project (HGP) spurred a great increase in
sequencing throughput and this, along with technical improvements,
resulted in a corresponding drop in sequencing costs. In contrast
to the 13 years and cost of nearly three billion US dollars, per
genome sequencing costs have been reduced significantly--indeed two
individual genomes have recently been completed (McGuire et al.,
Science 317:1687 (2007)). Personal genomes represent a paradigm
shift in medical treatment for both patients and health care
providers. By managing genetic risk factors for disease, health
care providers can more readily practice preventative medicine and
provide customized treatment. With large banks of completed
genomes, drug design and administration can be more efficient,
pushing forward the nascent field of pharmacogenomics.
[0003] Most conventional chemical or biochemical assays are based
on "bulk" measurements. In such measurements, a collective behavior
of a plurality of molecules within a certain volume of a sample
solution is measured to determine the properties of the molecules.
Recently, the detection of single molecule became possible.
Single-molecule detection provides another option for chemical and
biochemical assays, which offers much higher sensitivity and
provides more detailed information than conventional bulk
measurements, and soon became a new trend. An overview of the
criteria for achieving single-molecule detection is discussed in,
for example, the review articles by Moerner et al. (Moerner and
Fromm, "REVIEW ARTICLE: Methods of single-molecule fluorescence
spectroscopy and microscopy", Review of Scientific Instruments
74(8): 3597-3619 (2003)) and Walter et al. (Walter, et al.,
"Do-it-yourself guide: how to use the modern single-molecule
toolkit", Nature Methods 5: 475-489 (2008)). These articles also
discuss methods and apparatus that have been used or proposed for
single-molecule detection.
[0004] U.S. Pat. No. 7,170,050 provides a zero-mode waveguide (ZMW)
for single-molecule detection. The ZMW consists of a metal film and
a plurality of holes formed therein, which constitute the core
regions of the ZMW. In a ZMW, propagation of light having a
wavelength longer than a cutoff wavelength in a core region is
prohibited. When a light having a wavelength longer than the cutoff
wavelength is incident to the entrance of the waveguide, the light
will not propagate along the longitudinal direction of the core
region. Instead, the light intensity will decay exponentially along
the longitudinal direction of the core region, forming an
evanescent field at the entrance of the waveguide. This offers a
specific excitation zone, within which molecule is excited and the
emitted fluorescent light is captured by confocal microscope.
However, the detectable number of ZMWs is limited by the numerical
aperture (NA) of the confocal microscope and the throughput is
limited. U.S. Pat. No. 7,486,865 provides a recessed ZMW formed by
extending the ZMW into the underlying substrate. This configuration
allows a more tunable observation volume and higher signal level
for optics placed below the waveguide. However, this configuration
still has scale-up issue and limited throughput problems.
[0005] Therefore, there is a need for an apparatus to detect an
object, especially an object emitting light of low intensity such
as a single-molecule object.
SUMMARY
[0006] In accordance with the present disclosure, there is provided
an apparatus for detecting an object capable of emitting light. The
apparatus comprises a waveguide. The waveguide comprises a core
layer and a first cladding layer. At least one nanowell is formed
in at least the first cladding layer. The apparatus further
comprises a light detector. The light detector can detect a light
emitted from a single molecule object contained in the at least one
nanowell.
[0007] Also in accordance with the present disclosure, there is
provided a method of detecting a single molecule, comprising the
steps of: (a) emitting, by a light source, an incident light; (b)
coupling, by a coupler, the incident light into a waveguide,
forming an excitation light in the waveguide; (c) forming, by the
excitation light and at least one nanowell formed in at least a
cladding layer of the waveguide, an effective excitation zone; and
(d) exciting, by the excitation light, a single molecule object in
the effective excitation zone, to cause the single molecule object
to emit a light to be detected by a detector.
[0008] Also in accordance with the present disclosure, there is
provided a method of sequencing a nucleic acid, comprising the
steps of (a) providing a detection apparatus comprising: a
waveguide comprising: a core layer; and a first cladding layer; at
least one nanowell formed in at least the first cladding layer; and
a detector; (b) providing at least one nucleic acid molecule; (c)
locating the at least one nucleic acid molecule individually within
the at least one nanowell; (d) performing single molecule
sequencing-by-synthesis of the at least one nucleic acid molecule,
wherein the single molecule nucleic acid sequencing-by-synthesis
leads to emission of light correlated to the identity of at least
one base in the nucleic acid; (e) detecting the light with the
detector, resulting in an output signal; and (f) processing the
output signal to determine an identity of at least one base in the
nucleic acid.
[0009] Additional objects and advantages of the present disclosure
will be set forth in part in the description which follows, and in
part will be obvious from the description, or may be learned by
practice of the present disclosure. The objects and advantages of
the present disclosure will be realized and attained by means of
the elements and combinations particularly pointed out in the
appended claims.
[0010] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory and are not restrictive of the present disclosure,
as claimed.
[0011] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate one (several)
embodiment(s) of the present disclosure and together with the
description, serve to explain the principles of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a schematic view of a detection apparatus
consistent with the present disclosure.
[0013] FIG. 2 is a schematic view showing nanowells of different
sizes consistent with the present disclosure.
[0014] FIG. 3 is a schematic view showing different nanowell
designs consistent with the present disclosure.
[0015] FIG. 4 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0016] FIG. 5 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0017] FIG. 6 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0018] FIG. 7 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0019] FIG. 8 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0020] FIG. 9 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0021] FIG. 10 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0022] FIG. 11 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0023] FIG. 12 shows an absorbing-emitting light coupler consistent
with the present disclosure.
[0024] FIG. 13 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0025] FIG. 14 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0026] FIG. 15 is a schematic view showing a detection apparatus
according to one embodiment of the present disclosure.
[0027] FIGS. 16A-16D show the computer-simulation results for
different nanowells.
[0028] FIGS. 17A and 17B show the dependence of the transmittances
of TE and TM lights through a metal grating shown in FIG. 5 on the
grating period and depth of the metal grating. FIG. 17C shows the
ratio of the data shown in FIG. 17B to FIG. 17A.
[0029] FIGS. 18A and 18B show the dependence of the transmittance
of a light through a metal grating shown in FIG. 5 on the incident
angle of the light.
[0030] FIG. 19 shows a computer-simulation result for a detection
apparatus as shown in FIG. 20.
[0031] FIG. 20 schematically shows a exemplary detection apparatus
using a prism as a coupler.
[0032] FIG. 21 shows the simulated speckle of the incident light
coupled into the waveguide of a detection apparatus as shown in
FIG. 7.
[0033] FIGS. 22A and 22B show a lens used in a detection apparatus
for the simulation shown in FIG. 20.
[0034] FIG. 23 shows measured power of lights coupled into the
waveguide of a detection apparatus as shown in FIG. 9.
[0035] FIG. 24 shows a computer-simulation result for a detection
apparatus as shown in FIG. 8.
[0036] FIG. 25 shows a computer-simulation result for a detection
apparatus as shown in FIG. 9.
DETAILED DESCRIPTION
[0037] The apparatuses for conventional bulk assay, including
miniaturized bulk assay, and apparatuses for single-molecule
detection may share many essential elements and may have similar
apparatus structures. However, to realize single-molecule
detection, a system may need to fulfill at least the following two
criteria: 1) it should have both a confined excitation space and a
confined observation space, and 2) the above-noted two spaces
should fully or partially overlap and the overlapping region should
be small enough to ensure that the light emitted from the target
single molecule is higher than the background to provide a
detectable signal-to-noise ratio (SNR). For example, the volume of
the overlapping region should be on the order of or smaller than
femto-liter level. More particularly, the volume of the overlapping
region should be in the range from atto-liter to zepto-liter.
Moreover, it may also be important to prevent the excitation light
from reaching the detector.
[0038] Embodiments consistent with the present disclosure include a
detection apparatus and method of using the detection apparatus for
detecting an object, such as a single molecule object. The
detection apparatus is capable of detecting weak light emitted from
the object.
[0039] Hereinafter, embodiments consistent with the present
disclosure will be described in detail with reference to drawings.
Wherever possible, the same reference numbers will be used
throughout the drawings to refer to the same or like parts.
1. APPARATUS OF THE PRESENT DISCLOSURE
[0040] In one aspect, the disclosure relates to a detection
apparatus which is capable of detecting an object, such as a
single-molecule object. Consistent with the present disclosure, the
object may be a source of luminescence, such as a fluorescent dye
molecule, a phosphorescent dye molecule, a quantum dot, or a
light-emitting nanoparticle. The object may also be a
light-scattering particle. In addition, the object may be a target
molecule without light emitting capability, but may be attached to
a labeling object which is capable of emitting light (e.g., a
fluorescent dye molecule, a phosphorescent dye molecule, or a
quantum dot). A certain labeling object may be capable of being
attached to a specific target molecule. Thus, the target molecule
may be identified via the labeling object. More than one labeling
object may be attached to one target molecule.
1.1 Overview of the Apparatus
[0041] The detection apparatus consistent with the present
disclosure may comprise at least one light source, which can emit a
light, which may then be at least partially coupled into the
waveguide as an excitation light to excite the object. The light
source may be, for example, laser such as He--Ne laser and laser
diode (LD), light emitting diode (LED), organic light emitting
diode (OLED), quantum dot light emitting diode (OLED), fiber light,
or arc discharge fluorescent lamp.
[0042] The detection apparatus consistent with the present
disclosure may comprise a waveguide. The waveguide may be a channel
waveguide or a planar waveguide. The waveguide may comprise a core
layer and at least one cladding layer. For example, if the
waveguide is a channel waveguide, it may comprise a core layer and
a cladding layer surrounding the core layer. As another example, if
the waveguide is a planar waveguide, it may comprise a core layer
and one cladding layer arranged on the core layer or two cladding
layers sandwiching the core layer. The core layer has a larger
refractive index than the at least one cladding layer. The
excitation light may propagate in the core layer of the
waveguide.
[0043] Consistent with the present disclosure, at least one
nanowell may be formed in at least the at least one cladding layer.
The nanowell may comprise an upper opening and a bottom surface,
wherein the upper opening may be larger than the bottom surface.
The nanowell may extend through partial thickness of the at least
one cladding layer, full thickness of the at least one cladding
layer, full thickness of the at least one cladding layer and
partial thickness of the core layer, or the full thickness of the
at least one cladding layer and full thickness of the core layer.
An effective excitation zone may be formed near the bottom of the
nanowell. The lower boundary of the effective excitation zone may
be the bottom of the nanowell. The upper boundary of the effective
excitation zone may be defined by the distance to which the
excitation light can reach in the nanowell in the direction
perpendicular to the longitudinal direction of the core layer
(hereinafter, vertical direction).
[0044] The detection apparatus consistent with the present
disclosure may comprise a plurality of nanowells. Therefore, the
apparatus may also be used to monitor a large number of
objects.
[0045] The detection apparatus consistent with the present
disclosure may comprise a light detector detecting light emitted
from the object. Consistent with the present disclosure, the light
detector may comprise an optical sensor, which is capable of at
least partially absorbing light incident thereon and generating
output signals in response to the light. The optical sensor may be,
e.g., a p-n photodiode, a p-i-n photodiode, a multi-junction
photodiode, an avalanche photodiode (APD), a phototransistor, a
quantum-well infrared photodetector (QWIP), a photoconductive type
optical sensor, a photovoltaic type optical sensor, a thin-film on
ASIC (TFA), a metal-semiconductor-metal (MSM) photodetector, a
charge coupled device (CCD), a CMOS sensor, or a combination
thereof.
[0046] Consistent with the present disclosure, the light detector
may comprise a control circuit for controlling the operation of the
light detector. The control circuit may comprise a circuit of
signal amplifier, A/D convertor, integrator, comparator, logic
circuit, readout circuit, memory, microprocessor, clock, and/or
address.
[0047] Consistent with the present disclosure, the light detector
may be arranged at a place that the light emitted from the object
can reach. For example, the light detector may be arranged at the
opposite side of the core layer with respect to the nanowell. That
is, if the nanowell is arranged on one side of the core layer in
the vertical direction, the light detector may then be arranged on
the other side of the core layer in the vertical direction.
[0048] The detection apparatus consistent with the present
disclosure may comprise a light coupler. The light coupler may
couple at least part of the light emitted from the at least one
light source into the waveguide. The light coupler may be, e.g., a
prism coupler, a grating coupler, a side-injection coupler, a
vertical-injection coupler, or a co-directional coupler.
1.2 Exemplary Apparatuses
[0049] Referring to FIG. 1, a schematic view of a detection
apparatus 100 consistent with the present disclosure is
illustrated. In some embodiments, the detection apparatus 100 may
comprise a light source 102, a light coupler 104, a light detector
106, and a planar waveguide 110. The planar waveguide 110 may be
formed on a substrate (not shown). The light detector 106 may be
formed on or in the substrate.
[0050] The light source 102 may emit a light, which may be at least
partially coupled into the planar waveguide 110 by the light
coupler 104. Light coupled into the planar waveguide 110 may
propagate in the core layer of the planar waveguide 110 and serve
as the excitation light.
1.2.1 Waveguide
[0051] As shown in FIG. 1, in some embodiments, the planar
waveguide 110 may comprise a core layer 112, an upper cladding
layer 114, and a lower cladding layer 116. The core layer 112 may
comprise a material having a refractive index of n.sub.2, such as
silicon-titanium oxide (Si.sub.xTi.sub.1-xO.sub.2, where
0<x<1), titanium oxide, tantalum oxide, niobium oxide,
hafnium oxide, aluminum oxide, zirconium oxide, silicon nitride,
aluminum nitride, titanium nitride, polycarbonate (PC), PMMA, or
Su8. The upper and lower cladding layers 114 and 116 may comprise
materials having a refractive index of n.sub.3 and n.sub.4,
respectively. The materials for the upper and lower cladding layers
114 and 116 may be the same or may be different. Suitable material
for the upper cladding layer 114 or the lower cladding layer 116
may comprise, for example, silicon oxide, magnesium fluoride,
calcium fluoride, aluminum oxide, Su8, PMMA, or polycarbonate. The
refractive index n.sub.2 of the core layer 112 may be higher than
the refractive indices n.sub.3 and n.sub.4 of the upper and lower
cladding layers 114 and 116.
[0052] As noted above, for single molecule detection, one may need
to prevent the excitation light from reaching the detector. In a
planar waveguide, the surface of the core layer may not be as
smooth as would be desired. The rough surface of the core layer may
scatter part of the excitation light. It has been estimated that,
for a core layer having a surface roughness of about 0.3 nm, about
0.01% excitation light may be scattered and produce the noise. In
order to reduce the noise coming from surface scattering of
excitation light propagating within the core, the surface roughness
of the core should be less than about 0.3 nm.
1.2.2 Nanowell
[0053] In some embodiments, at least one nanowell 120 may be formed
in at least the upper cladding layer 114. The upper opening of the
nanowell 120 may be larger than the bottom of the nanowell 120. The
shape of the nanowell 120 is not limited. For example, the
horizontal cross section of the nanowell 120 may have a circular
shape, an oval shape, a rectangular shape, a square shape, or a
diamond shape. As shown in FIG. 2, the size of the bottom of the
nanowell 120 is also not limited. For example, the size of the
bottom of the nanowell 120 may be smaller than about the wavelength
of the excitation light. In some embodiments, the size of the
bottom of the nanowell 120 may be smaller than about one-half,
about one-quarter, or about one-eighth of the wavelength of the
excitation light. As used herein, "size" may refer to diameter,
length of the long axis, or length of the long side if the
horizontal cross section of the nanowell 120 has a circular shape,
an oval shape, or a rectangular shape. If the horizontal cross
section of the nanowell 120 has a square or a diamond shape, "size"
may refer to the length of the side. In one embodiment, the
diameter of the upper opening of the nanowell 120 may be about 1 to
about 10 .mu.m and the diameter of the bottom of the nanowell 120
may be about 10 to about 500 nm, the angle of the sidewall of the
nanowell relative to the direction perpendicular to the bottom of
the nanowell may be less than about 60 degree. Such a configuration
may ensure that one single molecule can enter a region near the
bottom of the nanowell 120 and be detected.
[0054] Consistent with the present disclosure, part of the
excitation light may enter the nanowell 120 and may, together with
the spatial confinement of the nanowell 120, form an effective
excitation zone 130. The effective excitation zone 130 may be
formed near the bottom of the nanowell 120. When an object enters
the effective excitation zone 130, it may be excited by the
excitation light and emit a light to be detected by the light
detector 106. Outside the effective excitation zone 130, an object
may not be excited by the excitation light, or its emitted light
cannot reach the light detector. It is to be understood that, the
dashed line in the figure schematically illustrates the approximate
upper boundary of the effective excitation zone 130, and does not
limit the shape of the upper boundary of the effective excitation
zone 130. For example, the upper boundary of an effective
excitation zone may be in a curved shape.
[0055] Depending on different conditions, such as the position of
the nanowell and/or the depth of the nanowell extending in the
waveguide, a different effective excitation zone may be formed. In
addition, the electromagnetic field in the effective excitation
zone may be, for example, an evanescent field, a mixture of
evanescent and travelling fields, or a travelling field, as
described in more detail below.
[0056] FIG. 3 schematically shows, as examples, different nanowell
designs consistent with the present disclosure. In some
embodiments, nanowell 121 may extend through partial thickness of
the upper cladding layer. In some embodiments, nanowell 122 may
extend through full thickness of the upper cladding layer. For
nanowell 121 or 122, when the excitation light propagates in the
core layer, although there may not be travelling light in the
nanowell 121 or 122, part of the light travelling in the core layer
may penetrate slightly into the nanowell 121 or 122. The light
penetrating into the nanowell 121 or 122 may decay exponentially in
the vertical direction, forming an evanescent field. This
evanescent field, together with the spatial confinement of the
nanowell 121 or 122, may form an effective excitation zone 131 or
132.
[0057] In some embodiments, nanowell 123 may extend through full
thickness of the upper cladding layer and partial thickness of the
core layer. For nanowell 123, besides an evanescent field, a
travelling field component may also appear in the nanowell, forming
an effective excitation zone 133.
[0058] In some embodiments, nanowell 124 may extend through full
thickness of the upper cladding layer and full thickness of the
core layer. For nanowell 124, most of the electromagnetic field in
the nanowell may be a travelling field, and an effective excitation
zone 134 is formed.
[0059] For a planar waveguide comprising nanowell 122, since the
bottom end of the nanowell is located right on the upper surface of
the core layer, the volume of the effective excitation zone 132 may
be equal to the effective region of the evanescent field, and may
be calculated approximately using the following equation:
V=.pi..times.(D/2).sup.2.times.h
where D is the diameter of the bottom of the nanowell and h is the
penetration depth of the evanescent field in the nanowell. For
example, if D and h are 100 nm and 100 nm, respectively, the
calculated volume of the effective excitation zone is approximately
1.times.10.sup.-18 liter, which equals to 1 atto-liter.
[0060] In some embodiments, the surfaces of the nanowell 120 and
the surface of the upper cladding layer 114 (the surface of the
upper protection layer, as described in the following, if one is
formed over the upper cladding layer) may possess different surface
properties. The surface properties may comprise, e.g.,
hydrophobicity, functional group, functional group density,
material density, or conductivity.
[0061] In some embodiments, the sidewall surface of the nanowell
120 may be hydrophilic, comprising a member chosen from silicon,
silica, metal, or metal oxide, and the bottom surface of the
nanowell 120 may be hydrophobic. However, if the bottom surface of
the nanowell 120 is made of a material with hydrophilic property,
it may be modified to be hydrophobic. For example, if the bottom
surface of the nanowell 120 is made of silicate or metal with
hydrophilic property, it may be modified to be hydrophobic using,
for example, R1.sub.x--Si(O--R2).sub.4-x (where R1 is a hydrophobic
group, such as alkyl chain --(CH.sub.2).sub.n--CH.sub.3, and R2 is
C.sub.nH.sub.2n+1, and where x is integer and 1.ltoreq.x.ltoreq.3
and n is an integer) or using, for example, polymers with a
functional group chosen from --COOH, --PO.sub.3H.sub.2, --SH, or
--NH.sub.2. As another example, if the bottom surface of the
nanowell 120 is made, of metal oxide with hydrophilic property, it
may be modified to be hydrophobic using, for example,
R1.sub.x-Si(O--R2).sub.4-x (where R1 is a hydrophobic group, such
as alkyl chain --(CH.sub.2).sub.n--CH.sub.3, and R2 is
C.sub.nH.sub.2n+1, and where x is integer and 1.ltoreq.x.ltoreq.3
and n is an integer) or using, for example, polymers with a
functional group chosen from --COOH, --PO.sub.3H.sub.2, --SH, or
NH.sub.2. By making the bottom surface of the nanowell 120
hydrophobic but keeping the sidewall surface of the nanowell 120
hydrophilic, the object being detected may be kept in the effective
excitation zone near the bottom of the nanowell 120 but may not
adhere to the sidewall surface of the nanowell 120. Thus, the
object may be effectively excited by the excitation light entering
the effective excitation zone.
[0062] In some embodiments, a plurality of nanowells may be formed
in the waveguide. In some embodiments, for each of the plurality of
nanowells, a light detector may be formed to detect the light
emitted from an object in the effective excitation zone of the
nanowell. In some embodiments, one light detector may be used to
detect the light emitted from objects in the effective excitation
zones of a plurality of nanowells.
1.2.3 Protection Layers
[0063] In some embodiments, protection layer(s) may be formed in
the detection apparatus to absorb scattered excitation light and/or
to block the ambient light from outside the detection apparatus, so
as to increase the signal-to-noise (S/N) ratio. Referring to FIG.
4, in some embodiments, an upper protection layer 142 and a lower
protection layer 144 may be formed over the upper cladding layer
114 and below the lower cladding layer 116, respectively. That is,
the upper protection layer 144 may be formed on the same side of
the waveguide as the nanowell 120, and the lower protection layer
144 may be formed on the opposite side of the waveguide and
arranged between the lower cladding layer 116 and the light
detector 106. In some embodiments, the detection apparatus may have
the upper protection layer 142 formed therein. In some embodiments,
the detection apparatus may have the lower protection layer 144
formed therein. In some embodiments, the detection apparatus may
have both upper and lower protection layers 142 and 144 formed
therein.
[0064] In some embodiments, the upper and lower protection layers
142 and 144 may be made of opaque material, such as metal or alloy.
The upper and lower protection layers 142 and 144 may be made of
the same material or be made of different materials. Suitable
material for upper and lower protection layers 142 and 144
comprises, for example, Al, Ti, Ni, Cr, Au, Cu, Pt, or Pd, or the
alloy of any two or more of them.
[0065] In some embodiments, a pinhole 150 may be formed in the
lower protection layer 144 at a position below the nanowell 120.
Light emitted from the object in the effective excitation zone 130
may pass through the pinhole and be detected by the light detector
106.
[0066] In some embodiments, as shown in FIG. 5, a nanostructured
metal pattern 152 functioning as a grating may be arranged in the
lower protection layer 144 instead of the pinhole. By properly
designing the pitch and depth of the metal pattern 152, most of the
light emitted from the object may pass through the metal pattern
152 but the noise originated from the excitation light may be
minimized.
[0067] The light emitted from the object may be a TM mode light and
the excitation light may be a TE mode light. The transmittance of a
TM mode light or a TE mode light through a metal grating may depend
on the pitch (i.e., grating period) and depth of the metal pattern
152. The transmittance may also depend on the refractive difference
between the metal and the material surrounding the metal. Moreover,
the transmittance may also depend on the angle of the light
incident on the metal grating. Therefore, the S/N ratio may be
further improved.
1.2.4 Light Coupler
[0068] Referring again to FIG. 1. A light coupler 104 may be
arranged near the waveguide or formed on or in the waveguide. The
light coupler 104 may be able to couple at least part of the
incident light from the light source 102 into the waveguide
110.
[0069] In some embodiments, a prism coupler may be used as the
light coupler 104. As schematically shown in FIG. 6, the prism
coupler may comprise a prism 202 and a collimating lens 204. The
incident light emitted from the light source 102 may be focused on
the same position of the waveguide 110 by the collimating lens 204.
As shown in FIG. 6, part of the incident light may be coupled into
the waveguide 110 by the prism coupler and propagate in the core
layer 112 as the excitation light. Depending on the position of the
light source and/or the incident angle of the incident light with
respect to the collimating lens 204, light with different modes may
be coupled into the waveguide 110 and propagate in the core layer
112, such as those shown by the dashed curves in FIG. 6. Therefore,
the mode of the excitation light propagating in the waveguide may
be adjustable and selectable. In some embodiments, the collimating
lens 204 may be specially designed so as to expand a point light
source into a linear light source, which may provide a
laterally-expanded excitation light to cover larger area in the
lateral direction, i.e., the direction perpendicular to the
cross-section shown in FIG. 6, of the waveguide 110.
[0070] In some embodiments, as schematically shown in FIG. 7, a
side coupler 302 may be used as the light coupler 104. The side
coupler 302 may be an optical lens module. The incident light may
be focused by the side coupler 302 onto and coupled into the
waveguide 110, and propagate in the core layer 112 as the
excitation light.
[0071] In some embodiments, a grating coupler may be used as the
light coupler 104. A grating coupler is an optical component with a
regular pattern, which may split and diffract light into several
beams propagating in different directions. Therefore, part of the
incident light may be guided into the waveguide 110 and propagate
in the core layer 112 of the waveguide 110.
[0072] In some embodiments, as schematically shown in FIG. 8, the
grating coupler may comprise a first grating 402 arranged at the
interface between the upper cladding layer 114 and the core layer
112. Due to the interference of the light reflected from the upper
and lower surface of the lower cladding layer 116, the coupling
efficiency of such a grating coupler may depend on the thickness of
the lower cladding layer 116.
[0073] As noted above, the incident light may not be totally
coupled into the core layer 112. Part of the incident light may
vertically pass through the waveguide 110 and be wasted. In some
embodiments, in order to improve the coupling efficiency, a
reflector (not shown) may be arranged below the waveguide 110. The
reflector may reflect the light passing through the lower cladding
layer 116 back to the core layer 112, causing it to be partially
coupled into the core layer 112, so as to increase the total amount
of light coupled into the core layer 112.
[0074] In some embodiments, the grating coupler may further
comprise a second grating 404 arranged at the interface between the
core layer 112 and the lower cladding layer 116, as shown in FIG.
9. The second grating 404 may be arranged right below the first
grating 402. In some embodiments, the coupling efficiency may be
further increased by adding more gratings. For example, if the
detection apparatus comprises upper and lower protection layers,
gratings may also be arranged at the interfaces between the
protection layers and the cladding layers, as schematically shown
in FIG. 10.
[0075] In some embodiments, a portion of the upper cladding layer
114 may be removed to expose the core layer 112. A grating, such as
grating 406 shown in FIG. 11, may be arranged on the exposed
portion of the core layer 112.
[0076] The shape of the grating may not be limited to that shown in
FIGS. 8-11. For example, to increase the coupling efficiency, in
some embodiments, a grating with curved structure may be used, such
as blaze grating or multi-level grating, where multi-level grating
simulates the blaze grating by dividing the blaze into several
steps.
[0077] In some embodiments, the light may also be indirectly
coupled into the waveguide 110 using an absorbing-emitting light
coupler. FIG. 12 schematically shows an absorbing-emitting light
coupler. The absorbing-emitting light coupler may comprise a
photoluminescent layer, which may absorb the incident light and
emit a fluorescent light with a longer wavelength. Light emitted by
the light source may be incident on the upper surface of the
absorbing-emitting light coupler, and the fluorescent light may be
emitted from the side of the absorbing-emitting light coupler. Part
of the fluorescent light may be coupled into the waveguide and
propagate in the core layer of the waveguide as the excitation
light to excite the object. The power coupling efficiency may be
calculated as follows:
P(.lamda..sub.2)/P.sub.0(.lamda..sub.1)=(1-T.sub.1)(1+R.sub.1T.sub.1).ti-
mes..phi..sub.FL.times.T.sub.2.times..eta..sub.c.times.T.sub.3
where .lamda..sub.1 and .lamda..sub.2 are the wavelengths of the
incident light and light emitted from the side of the
absorbing-emitting light coupler, respectively.
P.sub.0(.lamda..sub.1) and P(.lamda..sub.2) are the power of the
incident light and the power of the light coupled into the core
layer 112 of the waveguide 110, respectively. R.sub.1 is the
reflectivity at the interface between the absorbing-emitting light
coupler and the lower cladding layer 116. T.sub.1 is the
transmittance of a light with a wavelength of .lamda..sub.1 in the
absorbent layer after travelling a distance of d.sub.1 and T.sub.2
is the transmittance of a light with a wavelength of .lamda..sub.2
in the absorbent layer after travelling a distance of d.sub.2.
.phi..sub.FL is the photoluminescence quantum yield of the
photoluminescence material. .eta..sub.c is the coupling efficiency
of the absorbing-emitting light coupler. T.sub.3 is the
transmittance of the light emitted from the absorbing-emitting
light coupler passing through the waveguide with a total length of
d.sub.3.
[0078] For an absorbing-emitting light coupler, since the area of
the side surface is much smaller than the area of the upper
surface, the power intensity of the light emitted from the
absorbing-emitting light coupler and coupled into the core layer
112 may be much higher than that of the light incident on the
absorbing-emitting light coupler.
[0079] The photoluminescence material used in the
absorbing-emitting light coupler may be selected based on the
Stokes shift. For example, the photoluminescence material may have
a Stokes shift of equal to or larger than about 30 nm. In some
embodiments, the photoluminescence material may be
photoluminescence dyes, such as anthracene, coumarin, pyrene,
stilbene, porphyrin, perylene, Alq3, eosin, Bodipy dyes,
fluorescein, rhodamine, polymethine dye, DCM or its derivatives. In
some embodiments, the photoluminescence material may be
photoluminescence polymers, such as PPV or its derivatives. In some
embodiments, the photoluminescence material may be inorganic
material, such as quantum dots, alumina oxide, or zinc oxide.
[0080] In some embodiments, the coupling efficiency may be
increased by combining the grating with the absorbing-emitting
light coupler. The combined light coupler may comprise a grating
502 and an absorbing-emitting material 504. In some embodiments, as
shown in FIG. 13, the combined light coupler may be arranged on the
surface of an exposed portion of the core layer 112. In some
embodiments, as shown in FIG. 14, the combined light coupler may be
arranged within the core layer 112. The grating may help to
increase the path length of the incident light travelling in the
absorbing-emitting material, so as to increase the amount of light
being absorbed by the absorbing-emitting material and thus the
photoluminescence efficiency. Therefore, more incident light may be
converted to the excitation light and the power coupling efficiency
may be increased.
1.2.5 Other Optional Components of the Apparatus
[0081] In some embodiments, as shown in FIG. 15, an optical filter
160 may be arranged between the lower protection layer 144 and the
light detector 106. In some embodiments, the optical filter 160 may
be arranged between the lower cladding layer 116 and the detector
106 without the lower protection layer 144. In some embodiments,
the lower protection layer 144 itself may serve as an optical
filter. An optical filter may allow a light with a wavelength
within a certain range to pass through but at least partially block
a light with a wavelength outside the certain range. Therefore, by
properly choosing the optical filter 160, it may allow the light
emitted from the object to pass through but reduce the noise caused
by the excitation light, so as to improve the S/N ratio.
[0082] Consistent with the present disclosure, the object may be
contained in a sample solution, which may be filled in the nanowell
120. In some embodiments, a microfluidic channel (not shown) may be
used to conduct the sample solution into the nanowell 120. The
microfluidic channel may be designed in a way that the target
objects passes through the nanowell one at a time, so as to realize
a flow-cytometry-like detection. In some embodiments, a cover (not
shown) may be formed over the waveguide to hold the sample solution
and/or to block the ambient light.
[0083] In some of the above-described figures schematically showing
the structures of detection apparatuses consistent with the present
disclosure, for simplicity, some components are not shown. For
example, FIG. 6 shows the waveguide 110, the nanowell 120, and the
prism coupler comprising prism 202 and collimating lens 204. Other
components of the detection apparatus are not shown. It is to be
understood that the detection apparatuses shown in these figures
may also comprise other components as disclosed herein. For
example, the detection apparatus shown in FIG. 6 may also comprise
the light detector, the cover, the protection layer(s), and/or the
optical filter.
2. METHODS OF DETECTION AND APPLICATIONS OF THE PRESENT
DISCLOSURE
[0084] In another aspect, the disclosure relates to a method of
detecting an object, such as a single-molecule object, using the
detection apparatus as disclosed herein. Consistent with the
present disclosure, a sample solution comprising the object may be
filled in the nanowell 120 formed in the waveguide 110 of the
detection apparatus. An incident light emitted by a light source
may be partially coupled by a light coupler into the waveguide 110
and propagate in the core layer 112 of the waveguide 110. The light
coupled into the waveguide 110 may serve as an excitation light.
The object, when entering the effective excitation zone, may be
excited by the excitation light and emit a light to be detected by
a light detector.
[0085] The detection apparatuses and systems consistent with the
present disclosure, and method of using the same may be applied to,
e.g., nucleic acid detection, DNA sequencing, biomarker
identification, or flow cytometry. The detection apparatuses can
detect and process low intensity light signal, which makes single
molecule object detection possible.
2.1 Labels for Use with the Apparatus
[0086] In some embodiments of the methods of the present
disclosure, labels are attached to the analyte(s) (i.e., the
substance(s) to be detected), the probe(s), such as primers,
antibodies, or other reagents that interact with the analyte(s), or
other reagent(s), such as nucleotides (including nucleotide
analogs). Any label can be used on the analyte or probe which can
be useful in the correlation of signal with the amount or presence
of analyte.
[0087] For example, a wide variety of fluorescent molecules can be
utilized in the present disclosure including small molecules,
fluorescent proteins and quantum dots. Useful fluorescent molecules
(fluorophores) include, but are not limited to: 1,5 IAEDANS;
1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;
5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein;
5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM
(5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy
Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA
(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G;
6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);
7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine;
ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine);
Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;
Acriflavin Feulgen SITSA; AFPs-AutoFluorescent Protein-(Quantum
Biotechnologies); Texas Red; Texas Red-X conjugate;
Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;
Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole
Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3;
TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC
(TetramethylRodaminelsoThioCyanate); True Blue; Tru Red; Ultralite;
Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange;
Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; interchelating dyes
such as YOYO-3, Sybr Green, Thiazole orange; members of the Alexa
Fluor dye series (from Molecular Probes/Invitrogen) which cover a
broad spectrum and match the principal output wavelengths of common
excitation sources such as Alexa Fluor 350, Alexa Fluor 405, 430,
488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660,
680, 700, and 750; members of the Cy Dye fluorophore series (GE
Healthcare), also covering a wide spectrum such as Cy3, Cy3B,
Cy3.5, Cy5, Cy5.5, Cy7; members of the Oyster dye fluorophores
(Denovo Biolabels) such as Oyster-500, -550, -556, 645, 650, 656;
members of the DY-Labels series (Dyomics), for example, with maxima
of absorption that range from 418 nm (DY-415) to 844 nm (DY-831)
such as DY-415, -495, -505, -547, -548, -549, -550, -554, -555,
-556, -560, -590, -610, -615, -630, -631, -632, -633, -634, -635,
-636, -647, -648, -649, -650, -651, -652, -675, -676, -677, -680,
-681, -682, -700, -701, -730, -731, -732, -734, -750, -751, -752,
-776, -780, -781, -782, -831, -480XL, -481XL, -485XL, -510XL,
-520XL, -521 XL; members of the ATTO series of fluorescent labels
(ATTO-TEC GmbH) such as ATTO 390, 425, 465, 488, 495, 520, 532,
550, 565, 590, 594, 610, 611X, 620, 633, 635, 637, 647, 647N, 655,
680, 700, 725, 740; members of the CAL Fluor series or Quasar
series of dyes (Biosearch Technologies) such as CAL Fluor Gold 540,
CAL Fluor Orange 560, Quasar 570, CAL Fluor Red 590, CAL Fluor Red
610, CAL Fluor Red 635, Quasar 670; quantum dots, such as quantum
dots of the EviTags series (Evident Technologies) or quantum dots
of the Qdot series (Invitrogen) such as the Qdot 525, Qdot565,
Qdot585, Qdot605, Qdot655, Qdot705, Qdot 800; fluorescein;
rhodamine; and/or phycoerythrin; or combinations thereof. See,
e.g., U.S. Application Publication 2008/0081769.
[0088] In some embodiments, at least one bioluminescent or
chemiluminescent system is provided which generates light in the
presence of an entity such as an analyte, reagent, or reaction
product. For example, a bioluminescent or chemiluminescent system
can be used to detect pyrophosphate generated in a sequencing by
synthesis reaction (discussed in more detail below); to detect the
presence of metals such as iron or copper by their catalysis of a
light-generating reaction; or to measure the amount of a reagent
bound by an analyte, wherein the reagent comprises at least one
component of the bio- or chemi-luminescent system.
[0089] Examples of bioluminescent systems known in the art include
systems comprising at least one luciferase, e.g., firefly
luciferases, including Photinus pyralis luciferase. A
bioluminescent system can be used to detect pyrophosphate, for
example, by providing luciferase, ATP sulfurylase, luciferin, and
adenosine 5' phosphosulfate, together with the components of the
sequencing by synthesis reaction (in which dATP can be substituted
with an analog such as dATP.alpha.S to avoid nonspecific light due
to consumption of dATP by luciferase). When pyrophosphate is
generated by a nucleotide incorporation event, ATP sulfurylase
produces ATP in an adenosine 5' phosphosulfate dependent manner.
The ATP drives conversion of luciferin to oxyluciferin plus light
by luciferase. Other bioluminescent systems include systems based
on photoproteins such as aequorin, which oxidizes coelenterazine to
excited coelenteramide, which emits light.
[0090] Examples of chemiluminescent systems include luminol plus
hydrogen peroxide, which can undergo a light-emitting reaction in
the presence of a metal catalyst or auxiliary oxidant; diphenyl
oxalate plus hydrogen peroxide and a suitable dye, which undergoes
excitation and light emission in a multistep reaction that produces
carbon dioxide (examples of suitable dyes include phenylated
anthracene derivatives such as 9,10-diphenylanthracene,
9,10-Bis(phenylethynyl)anthracene, and
1-Chloro-9,10-bis(phenylethynyl)anthracene, and rhodamines such as
rhodamine 6G and rhodamine B); singlet oxygen-producing systems
such as hydrogen peroxide plus sodium hypochlorite; and systems
comprising an enzyme such as horseradish peroxidase, which acts on
luminol or other commercially available substrates.
[0091] In some embodiments, the methods of the present disclosure
comprise forming covalent attachments, such as between reagents or
analytes and labels, or between a reagent, such as a polymerase
used in a sequencing reaction, and a surface, such as the surface
of a nanowell. Many methods for forming covalent attachments, such
as of reagents to labels and/or surfaces, are known in the art.
Non-covalent attachment methods can also be used. A number of
different chemical modifiers can be used to facilitate attachment
formation. Examples of chemical modifiers include N-hydroxy
succinimide (NHS) groups, amines, aldehydes, epoxides, carboxyl
groups, hydroxyl groups, hydrazides, hydrophobic groups, membranes,
maleimides, biotin, streptavidin, thiol groups, nickel chelates,
photoreactive groups, boron groups, thioesters, cysteines,
disulfide groups, alkyl and acyl halide groups, glutathiones,
maltoses, azides, phosphates, and phosphines. In some embodiments,
attachments are formed between two entities by using an appropriate
combination of modifiers (e.g., an electrophilic modifier and a
nucleophilic modifier), wherein each entity comprises at least one
modifier.
[0092] In some embodiments, attachments are formed between two
entities by using a chemical modifier present on one of the
entities and a naturally occurring moiety, for example, an amine or
sulfhydryl, of the other entity. In some embodiments, modifiers
that are reactive to amines are used. An advantage of this reaction
is that it can be fast and can avoid production of toxic
by-products. Examples of such modifiers include NHS-esters,
aldehydes, epoxides, acyl halides, and thio-esters. Most proteins,
peptides, glycopeptides, etc., have free amine groups, which can
react with such modifiers to link them covalently to these
modifiers. Nucleic acid probes with internal or terminal amine
groups can also be synthesized, and are commercially available
(e.g., from IDT or Operon). Thus, biomolecules can be bound (e.g.,
covalently or non-covalently) to labels or other reagents using
similar chemistries.
[0093] A number of other multi-functional cross-linking agents can
be used to convert the chemical reactivity of one kind of modifier
to another. These groups can be bifunctional, tri-functional,
tetra-functional, and so on. They can also be homo-functional or
hetero-functional. An example of a bi-functional cross-linker is
X-Y-Z, where X and Z are two reactive groups, and Y is a connecting
linker. Further, if X and Z are the same group, such as NHS-esters,
the resulting cross-linker, NHS-Y-NHS, is a homo-bi-functional
cross-linker and could connect two entities that each comprise an
amine. If X is NHS-ester and Z is a maleimide group, the resulting
cross-linker, NHS-Y-maleimide, is a hetero-bi-functional
cross-linker and could link an entity comprising an amine with an
entity comprising a thio-group. Cross-linkers with a number of
different functional groups are widely available. Examples of such
functional groups include NHS-esters, thio-esters, alkyl halides,
acyl halides (e.g., iodoacetamide), thiols, amines, cysteines,
histidines, di-sulfides, maleimide, cis-diols, boronic acid,
hydroxamic acid, azides, hydrazines, phosphines, photoreactive
groups (e.g., anthraquinone, benzophenone), acrylamide (e.g.,
acrydite), affinity groups (e.g., biotin, streptavidin, maltose,
maltose binding protein, glutathione, glutathione-S-transferase),
aldehydes, ketones, carboxylic acids, phosphates, hydrophobic
groups (e.g., phenyl, cholesterol), etc.
[0094] Other modifier alternatives (such as photo-crosslinking and
thermal crosslinking) are known to those skilled in the art.
Commercially available technologies include, for example, those
from Mosiac Technologies (Waltham, Mass.), EXIQON.TM. (Vedbaek,
Denmark), Schleicher and Schuell (Keene, N.H.), Surmodics.TM. (St.
Paul, Minn.), XENOPORE.TM. (Hawthorne, N.J.), Pamgene
(Netherlands), Eppendorf (Germany), Prolinx (Bothell, Wash.),
Spectral Genomics (Houston, Tex.), and COMBIMATRIX.TM. (Bothell,
Wash.).
2.2 Nucleic Acid Detection
[0095] A detection apparatus consistent with the present disclosure
may be used as part of a system for or in methods or processes of
molecule detection, e.g., nucleic acid sequencing. This apparatus,
and methods or processes utilizing it, are useful for, e.g.,
analytical and diagnostic applications. These applications may be
private, public, commercial, or industrial.
[0096] A detection apparatus consistent with the present disclosure
may be used with a wide variety of sequencing modalities and may be
suitable for sequencing single molecules. Additionally, the
detection apparatus consistent with the present disclosure have
simplified design, assembly, and production relative to existing
biochip devices.
[0097] A detection apparatus consistent with the present disclosure
may be used as part of a system for or in methods and processes of
biomolecule detection, including nucleic acid hybridization or
sequencing for, e.g., whole genome sequencing, transcriptional
profiling, comparative transcriptional profiling, or gene
identification. Biomolecule detection can also include detection
and/or measurement of binding interactions, e.g., protein/protein,
antibody/antigen, receptor/ligand, and nucleic acid/protein. These
applications are useful for analytical or diagnostic processes and
methods.
2.2.1 Molecules to be Detected
[0098] Nucleic acids suitable for detection by the methods provided
by the present disclosure may include any nucleic acid, including,
for example, DNA, RNA, or PNA (peptide nucleic acid), and may
contain any sequence--both known and unknown, including naturally
occurring or artificial sequences. The nucleic acid may be
naturally derived, recombinantly produced, or chemically
synthesized. The nucleic acid may comprise naturally-occurring
nucleotides, nucleotide analogs not existing in nature, or modified
nucleotides. The length of the nucleic acid to be detected may vary
based on the actual application. In some embodiments, the nucleic
acid may include at least 10, 20, 50, 100, 200, 500, 1000, 2000,
5000, 10000, 20000 bases, or more. In some embodiments, the nucleic
acid may be from 10 to 20, from 10 to 50, from 10 to 100, from 50
to 100, from 50 to 500, from 50 to 1000, from 50 to 5000, from 500
to 2000, from 500 to 5000, or from 1000 to 5000 bases.
[0099] A nucleic acid may be single-stranded for detection. Single
stranded nucleic acid templates may be derived from a double
stranded molecule by means known in the art including, for example,
heating or alkali or other chemical treatment. Single stranded
nucleic acid templates may also be produced by, e.g., chemical or
in vitro synthesis.
[0100] In some embodiments, the nucleic acid to be detected may be
circular. In some embodiments, the methods of the present
disclosure comprise providing a circular nucleic acid molecule
comprising an insert with a known sequence, which can be used as a
binding site for a primer. The circular nucleic acid molecule can
be provided in a single or double stranded state, and will
generally comprise at least one covalently closed strand. Double
stranded circular molecules may comprise a nicked strand or a
second covalently closed strand.
[0101] In some embodiments, the circular nucleic acid molecule is
provided by isolating it in circular form from its source, if part
of its sequence is known and thus can serve as the nucleic acid
insert (e.g., a conserved motif within the sequence of a gene
contained in the circular molecule may be known, or the molecule
may be known to contain a sequence based on its ability to
hybridize under high stringency conditions to another nucleic acid
of known sequence). In some embodiments, the sequence of the
nucleic acid insert is known only inexactly, as would be the case
when knowledge of the sequence is derived from stringent
hybridization properties. In some embodiments, the sequence of the
nucleic acid insert is known exactly, such as would be the case
when the circular nucleic acid molecule has a known backbone
sequence or has been engineered to contain a known sequence.
[0102] In some embodiments, the circular nucleic acid molecule is
provided by performing an in vitro reaction or reactions to
incorporate a linear nucleic acid sample into a circular molecule
along with a nucleic acid insert. The in vitro reaction or
reactions can in some embodiments comprise ligation by a ligase
and/or other strand joining reactions such as can be catalyzed by
various enzymes, including recombinases and topoisomerases. DNA
ligase or RNA ligase may be used to enzymatically join the two ends
of a linear template, with or without an adapter molecule or
linkers, to form a circle. For example, T4 RNA ligase couples
single-stranded DNA or RNA, as described in Tessier et al., Anal
Biochem, 158: 171-78 (1986). CIRCLIGASE.TM. (Epicentre, Madison,
Wis.) may also be used to catalyze the ligation of a single
stranded nucleic acid. Alternatively, a double stranded ligase,
such as E. coli or T4 DNA ligase, may be used to perform the
circularization reaction.
[0103] In some embodiments, providing the circular nucleic acid
molecule comprises replicating a nucleic acid template by extending
from at least one primer (which can include random primers with 5'
flaps of known sequence that can serve as the nucleic acid insert)
comprising complementary regions and circularizing the amplified
nucleic acid, such as may be catalyzed by a ligase or a
recombinase; the amplified nucleic acid may in some embodiments be
processed at its ends, e.g., by restriction or phosphorylation,
prior to circularization.
[0104] In some embodiments, the circular nucleic acid molecule is
provided by performing chemical circularization. Chemical methods
employ known coupling agents such as BrCN plus imidazole and a
divalent metal, N-cyanoimidazole with ZnCl.sub.2,
1-(3-dimethylaminopropyl)-3 ethylcarbodiimide HCl, and other
carbodiimides and carbonyl diimidazoles. The ends of a linear
template may also be joined by condensing a 5'-phosphate and a
3'-hydroxyl, or a 5'-hydroxyl and a 3'-phosphate.
[0105] In some embodiments, the circular nucleic acid molecule
contains an insert sequence that could be considered an end link
primer (discussed below) except that it is not at an end, since the
molecule is circular.
2.2.1.1 End Link Primer
[0106] In some embodiments, a linear nucleic acid may further
comprise one or more end link primers coupled to the 5' end, the 3'
end, or both the 5' end and the 3' end of the nucleic acid. In
particular embodiments, an end link primer may be affixed to the 3'
end of the nucleic acid. End link primers may be used to provide a
complementary sequence for one or more detecting primers, e.g., a
sequencing primer.
[0107] End link primers are short nucleic acid molecules usually
composed of less than 100 nucleotides. In some embodiments, the end
link primer may be at least 5, 10, 15, 20, 25, 30, 50, 75, 90
nucleotides, or more, in length. In certain embodiments, end link
primers may be from 8 to 25, from 10 to 20, from 10 to 30, or from
10 to 50 nucleotides in length. In some embodiments, the end link
primers may be unbranched, however, in other embodiments, they may
be branched.
[0108] The end link primer may serve as a complement to one or more
primers used to detect the nucleic acid, e.g., a sequencing primer.
In some embodiments, the primer may be used to detect the nucleic
acid by hybridization, e.g., the primer may contain a detectable
label, e.g., a fluorescent label. In some embodiments, the 5' end
of the end link primer may comprise a sequence complementary to a
sequencing primer. In some embodiments, the end link primer
sequence that is complementary to the sequencing primer may be
oriented so that the 3' end of the sequencing primer may be
immediately adjacent to the first nucleotide in the nucleic acid to
be sequenced.
[0109] In some embodiments, end link primers may be added to ends
of the nucleic acid to be detected by a ligase, for example, a DNA
ligase. In some embodiments, the end link primer and nucleic acid
to be detected may be both single stranded before the ligation. In
other embodiments, both may be double stranded. In still other
embodiments, one may be single stranded and the other may be double
stranded. Ligation is well known in the art. For example, in the
polony sequencing method, Shendure et al. (Science, 309:1728-1732
(2005)) ligated a T30 end link primer (32 bp) to a sample DNA
segment with the New England Biolabs' (NEB) Quick Ligation kit.
There, the ligation reaction solution included 026 pMole of DNA,
0.8 pMole of T30 end link primer, 4.0 .mu.l T4 DNA Ligase, in
1.times. Quick Ligation Buffer. After mixing, the reaction solution
was incubated for about 10 minutes at room temperature, and then
placed on ice. The ligation reaction was stopped by heating the
samples to 65.degree. C. for 10 minutes.
[0110] In other embodiments, the end link primer may be synthesized
on the nucleic acid to be detected. For example, the end link
primer may be a homopolymer added by, e.g., terminal transferase.
For example, Harris et al., (Science 320:106-109 (2008)) added a
poly A tail to DNA templates, which served as the complement to a
poly T sequencing primer in the single molecule sequencing of a
viral genome.
2.2.1.2 Sequencing Primer
[0111] A sequencing primer is a single-stranded oligonucleotide
complementary to a segment of the nucleic acid to be detected or
its associated end link primer. In some embodiments, the sequencing
primer may be at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50
nucleotides, or more in length. In particular embodiments, the
sequencing primer may be from 8 to 25, from 10 to 20, from 10 to
30, or from 10 to 50 nucleotides in length. The sequencing primer
may be made up of any type of nucleotide, including
naturally-occurring nucleotides, nucleotide analogs not existing in
nature, or modified nucleotides.
[0112] In some embodiments, a sequencing primer may contain
modified nucleotides, e.g., locked nucleic acids (LNAs; modified
ribonucleotides, which provide enhanced base stacking interactions
in a polynucleic acid). As an illustration of the utility of LNAs,
Levin et al. (Nucleic Acid Research 34(20):142 (2006)) showed that
a LNA-containing primer had improved specificity and exhibited
stronger binding relative to the corresponding unlocked primer.
Three variants of the MCP1 primer (5'-cttaaattttcttgaat-3')
containing 3 LNA nucleotides (in caps) at different positions in
the primer were made: MCP1-LNA-3'(5'-cttaaattttCtTgaAt-3');
MCP1-LNA-5' (5'-CtTaAattttcttgaat-3'); and MCP1-LNA-even
(5'-ctTaaatTttctTgaat-3'). All LNA-substituted primers had enhanced
Tm, while the MCP1-LNA-5' primer exhibited particularly enhanced
sequencing accuracy (Phred 030 counts). Accordingly, in particular
embodiments, the sequencing primer may contain at least one locked
nucleotide in its 5' region, i.e., the 5' half, third, or quarter
of the sequencing primer.
[0113] Sequencing primers and single stranded sample nucleic acids
(i.e., a nucleic acid to be detected including at least one end
link primer) may be hybridized before being applied to a detection
apparatus consistent with the present disclosure. The sequencing
primer and sample nucleic acid may be hybridized by mixing the
sample nucleic acid with a molar excess of sequencing primer in a
salt-containing solution, such as 5.times.SSC (or 5.times.SSPE),
0.1% Tween 20 (or 0.1% SDS), and 0.1% BSA buffer. The mixture may
be heated to 65.degree. C. for at least 5 minutes and slowly cooled
to room temperature, to allow primer/template annealing. Residual
primers may be eliminated by appropriate means including, e.g., a
molecular sieve.
[0114] Primers, including both end link and sequencing primers, may
be designed by appropriate means, including visual inspection of
the sequence or computer-assisted primer design. Numerous software
packages are available to assist in the primer design, including
DNAStar.TM. (DNAStar, Inc., Madison, Wis.), OLIGO 4.0 (National
Biosciences, Inc.), Vector NTI.RTM. (Invitrogen), Primer Premier 5
(Premierbiosoft), and Primer3 (Whitehead Institute for Biomedical
Research, Cambridge, Mass.). Primers may be designed taking into
account, for example, the molecule to be sequenced, specificity,
length, desired melting temperature, secondary structure, primer
dimers, GC content, pH and ionic strength of the buffer solution,
and the enzyme used (i.e., polymerase or ligase). See, e.g., Joseph
Sambrook and David Russell, Molecular Cloning: A Laboratory Manual
Cold Spring Harbor Laboratory Press; 3rd edition (2001).
2.2.2 Sequencing Modalities
[0115] Some embodiments of the present disclosure are methods of
sequencing a nucleic acid, comprising the steps of (a) providing a
detection apparatus comprising: a waveguide comprising: a core
layer; and a first cladding layer; at least one nanowell formed in
at least the first cladding layer; and a detector; (b) providing at
least one nucleic acid molecule; (c) locating the at least one
nucleic acid molecule individually within the at least one
nanowell; (d) performing single molecule sequencing-by-synthesis of
the at least one nucleic acid molecule, wherein the single molecule
nucleic acid sequencing-by-synthesis leads to emission of light
correlated to the identity of at least one base in the nucleic
acid; (e) detecting the light with the detector, resulting in an
output signal; and (f) processing the output signal to determine an
identity of at least one base in the nucleic acid.
[0116] In these methods, "locating the at least one nucleic acid
molecule individually within the at least one nanowell" is
understood to mean that a single nucleic acid molecule is located
in a nanowell, i.e., there is at least one nanowell in which one
(and not more than one) nucleic acid molecule is located. In some
embodiments, there are a plurality of nanowells which each
individually contains one (and not more than one) nucleic acid
molecule. In some embodiments, during operation, some of the
plurality of nanowells contain nucleic acid molecules and others do
not. That is, the concentration of nucleic acid molecules in the
sample solution is lower than a certain value so that not all
nanowells have nucleic acid molecules contained in them. This may
prevent the scenario that two or more molecules enter the same
nanowell successively before a sequencing is completed, so as to
prevent the results of one sequencing from comprising information
from more than one molecules. For example, in some embodiments of
the present disclosure, less than or equal to 50%, 40%, 30%, 25%,
20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the
nanowells will generate a signal due to the low concentration of
the biological molecules to be detected or identified.
[0117] In some embodiments, the concentration of the nucleic acid
molecules in the sample solution may depend on the volume of the
effective excitation zone. For example, if the volume of the
effective excitation zone is 1 atto-liter, the concentration of the
nucleic acid molecules in the sample solution may be about 1.6
.mu.M. In some embodiments, the optimum concentration of the
nucleic acid molecules in the sample solution may be from about
1-100 .mu.M to about 1-100 .mu.M.
[0118] In some embodiments, the nucleic acid molecules to be
detected are provided at a concentration that is substoichiometric
relative to the volume of the effective excitation zone of the
nanowells of an apparatus according to the disclosure. For example,
if the effective excitation zone is 1 atto-liter, nucleic acid
molecules can be provided at a concentration ranging from 0.01 to
0.5 molecules per atto-liter, 0.05 to 0.2 molecules per atto-liter,
or about 0.1 molecules per atto-liter. The concentrations can be
scaled appropriately based on the effective excitation zone size.
In some applications, it may be desirable to use higher or lower
concentrations based on factors such as the relative importance of
minimizing multiple signals from the same nanowell versus
generating signal from a larger proportion of nanowells.
[0119] In some embodiments, the nucleic acid molecules to be
detected are provided at a concentration that is in stoichiometric
equivalence or excess relative to the volume of the effective
excitation zone of the nanowells of an apparatus according to the
disclosure. For example, if the effective excitation zone is 1
atto-liter, nucleic acid molecules can be provided at a
concentration ranging from 1 to 50 molecules per atto-liter, 2 to
20 molecules per atto-liter, or 3 to 10 molecules per atto-liter.
Use of these concentration ranges can be paired with provision of
the enzyme for sequencing the nucleic acid molecules at a
substoichiometric level, for example, 0.01 to 0.5 active,
accessible polymerases per nanowell, 0.05 to 0.2 active, accessible
polymerases per nanowell, or about 0.1 active, accessible
polymerases per nanowell.
[0120] Attachment of enzymes to be used for sequencing or other
detection reactions to a surface of a nanowell can result in some
enzymes being rendered inaccessible, inactive, or both due to
factors such as the location of the attachment and whether the
structure of the enzyme is affected. The number of active,
accessible polymerases per nanowell can be estimated empirically by
providing the other components of a positive control
sequencing-by-synthesis reaction in excess and observing how many
nanowells generate fluorescent signals consistent with the presence
of active, accessible enzyme. When a large fraction, such as 50% or
more of the nanowells, generate fluorescent signal, a random
distribution model would estimate that many of the nanowells
contain at least two active, accessible enzymes (e.g., when 50% of
the wells generate signal, about 25% of the wells are expected to
contain at least two active, accessible enzymes). In such a
situation, it can be advisable to limit the concentration of the
nucleic acid molecule to be sequenced in order to minimize the
frequency of two different sequencing complexes forming in the same
nanowell. Alternatively, when a small fraction, such as 10% or less
of the nanowells, generate fluorescent signal, a random
distribution model would estimate that few of the nanowells contain
at least two active, accessible enzymes (e.g., when 10% of the
wells generate signal, about 1% of the wells are expected to
contain at least two active, accessible enzymes). In such a
situation, it can be advisable to use a relatively high
concentration of the nucleic acid molecule to be sequenced in order
to minimize the frequency of no sequencing complexes forming in a
nanowell that does have an accessible, active enzyme.
[0121] In some embodiments, the single molecule nucleic acid
sequencing-by-synthesis leads to emission of light via
chemiluminescence. Notably, in these embodiments, it is not
necessary for the apparatus to comprise a light source, as
chemiluminescence generates light from chemical energy.
[0122] In some embodiments, the apparatus further comprises a light
source, which may be used to provide excitatory light, e.g., for
causing the single molecule nucleic acid sequencing-by-synthesis to
emit light via fluorescence.
[0123] The detection apparatuses and methods provided by the
present disclosure may be used to detect and sequence nucleic acids
by means known in the art, as reviewed in, e.g., U.S. Pat. No.
6,946,249 and Shendure et al., Nat. Rev. Genet. 5:335-44 (2004).
The sequence modalities can be chosen from single molecule
sequencing methods known in the art. In some embodiments, the
sequencing methods may rely on the specificity of either a DNA
polymerase or DNA ligase and may include, e.g., base extension
sequencing (single base stepwise extensions) and multi-base
sequencing by synthesis (including, e.g., sequencing with
terminally-labeled nucleotides). The methods typically involve
providing a sample nucleic acid, which may include at least one end
link primer. The nucleic acid may be provided in single stranded
form or may be rendered single stranded, e.g., by chemical or
thermal denaturation. Sequencing may be then initiated at a
sequencing primer.
[0124] In some embodiments, the methods of the present disclosure
comprise forming covalent attachments, such as between reagents or
analytes and surfaces or labels. For example, in single molecule
sequencing procedures, a nucleic acid molecule or an enzyme such as
a polymerase may be attached to the bottom of the nanowell. Such an
attachment can allow the acquisition of data over multiple
sequencing cycles. Many methods for forming covalent attachments,
such as of reagents to surfaces or labels, are known in the art.
Non-covalent attachment methods can also be used. A number of
different chemical modifiers can be used to facilitate attachment
formation. Examples of chemical modifiers include N-hydroxy
succinimide (NHS) groups, amines, aldehydes, epoxides, carboxyl
groups, hydroxyl groups, hydrazides, hydrophobic groups, membranes,
maleimides, biotin, streptavidin, thiol groups, nickel chelates,
photoreactive groups, boron groups, thioesters, cysteines,
disulfide groups, alkyl and acyl halide groups, glutathiones,
maltoses, azides, phosphates, and phosphines. These can easily be
prepared, for example, using standard methods (Microarray Biochip
Technologies, Mark Schena, Editor, March 2000, Biotechniques
Books). In some embodiments, attachments are formed between two
entities by using an appropriate combination of modifiers (e.g., an
electrophilic modifier and a nucleophilic modifier), wherein each
entity comprises at least one modifier.
[0125] In some embodiments, attachments are formed between two
entities by using a chemical modifier present on one of the
entities and a naturally occurring moiety, for example, an amine or
sulfhydryl, of the other entity. In some embodiments, modifiers
that are reactive to amines are used. An advantage of this reaction
is that it can be fast and can avoid production of toxic
by-products. Examples of such modifiers include NHS-esters,
aldehydes, epoxides, acyl halides, and thio-esters. Most proteins,
peptides, glycopeptides, etc., have free amine groups, which can
react with such modifiers to link them covalently to these
modifiers. Nucleic acid probes with internal or terminal amine
groups can also be synthesized, and are commercially available
(e.g., from IDT or Operon). Thus, biomolecules can be bound (e.g.,
covalently or non-covalently) to labels, surfaces, or other
reagents using similar chemistries.
[0126] A number of other multi-functional cross-linking agents can
be used to convert the chemical reactivity of one kind of modifier
to another. These groups can be bifunctional, tri-functional,
tetra-functional, and so on. They can also be homo-functional or
hetero-functional. An example of a bi-functional cross-linker is
X-Y-Z, where X and Z are two reactive groups, and Y is a connecting
linker. Further, if X and Z are the same group, such as NHS-esters,
the resulting cross-linker, NHS-Y-NHS, is a homo-bi-functional
cross-linker and could connect two entities that each comprise an
amine. If X is NHS-ester and Z is a maleimide group, the resulting
cross-linker, NHS-Y-maleimide, is a hetero-bi-functional
cross-linker and could link an entity comprising an amine with an
entity comprising a thio-group. Cross-linkers with a number of
different functional groups are widely available. Examples of such
functional groups include NHS-esters, thio-esters, alkyl halides,
acyl halides (e.g., iodoacetamide), thiols, amines, cysteines,
histidines, di-sulfides, maleimide, cis-diols, boronic acid,
hydroxamic acid, azides, hydrazines, phosphines, photoreactive
groups (e.g., anthraquinone, benzophenone), acrylamide (e.g.,
acrydite), affinity groups (e.g., biotin, streptavidin, maltose,
maltose binding protein, glutathione, glutathione-S-transferase),
aldehydes, ketones, carboxylic acids, phosphates, hydrophobic
groups (e.g., phenyl, cholesterol), etc.
[0127] Other modifier alternatives (such as photo-crosslinking and
thermal crosslinking) are known to those skilled in the art.
Commercially available technologies include, for example, those
from Mosiac Technologies (Waltham, Mass.), EXIQON.TM. (Vedbaek,
Denmark), Schleicher and Schuell (Keene, N.H.), Surmodics.TM. (St.
Paul, Minn.), XENOPORE.TM. (Hawthorne, N.J.), Pamgene
(Netherlands), Eppendorf (Germany), Prolinx (Bothell, Wash.),
Spectral Genomics (Houston, Tex.), and COMBIMATRIX.TM. (Bothell,
Wash.).
[0128] For single molecule sequencing modalities, the present
disclosure can offer the advantage of being able to resequence
single molecules. For example, a polymerase can be attached to a
nanowell surface, such as at the bottom. A template nucleic acid
molecule to be sequenced can be provided in circular form together
with a sequencing primer. Resequencing can be achieved by
performing a plurality of sequencing cycles such that a sequence
read is obtained that is greater than the number of nucleotides in
the template nucleic acid molecule. The sequencing read therefore
comprises information that redundantly identifies the base in at
least one position in the template nucleic acid molecule. In some
embodiments, the sequencing read comprises information that
redundantly identifies at least 25%, 50%, 75%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% of the bases in the template
nucleic acid molecule. In some embodiments, the sequencing read
comprises information that identifies at least 25%, 50%, 75%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the bases
in the template nucleic acid molecule with three-fold, four-fold,
five-fold, seven-fold, or ten-fold redundancy. By resequencing the
same molecule, sequencing errors are expected to fall as the power
of the number of sequencing reads. For example, if per base error
rates for a single read are 10.sup.-3, then after two reads, this
falls to (10.sup.-3).sup.2, i.e., 10.sup.-6. This is particularly
advantageous for single molecule sequencing since the modified
nucleotides used for sequencing can lose their labels or blocking
groups resulting in, e.g., spurious deletions.
[0129] In general, in single molecule sequencing, at least one
nucleic acid molecule to be sequenced is contacted with a primer.
The primer is modified, e.g., by performing at least one
enzyme-catalyzed polymerization or ligation reaction. The at least
one reaction leads to emission of light correlated to the identity
of at least one base in the nucleic acid. "Leading to" emission of
light is understood to mean that the at least one reaction causes
at least one condition under which light emission correlated to the
identity of at least one base in the nucleic acid occurs; this
occurrence may be via interaction with excitatory light, a chemi-
or bio-luminescent system, etc. The at least one condition can be,
for example, incorporation of a fluorophore into the product of the
at least one reaction, or the release of pyrophosphate. Thus, light
may be generated with or without external excitation. For example,
single molecule sequencing can be performed with reversible
terminator base analogs comprising a covalently-linked detectable
label, e.g., a fluorescent label, and a blocking group to prevent
any secondary extension, wherein the analog is excited and detected
after it has been added to the primer, and the label and blocking
group are removed after addition to allow another round of
extension. Alternatively, a product of an extension step, such as a
pyrophosphate, can be detected without external excitation by
providing a chemi- or bio-luminescent detection system which emits
light in a pyrophosphate-dependent manner. These and other
modalities are discussed in more detail below.
[0130] The light emitted is correlated to the identity of at least
one base in the nucleic acid. In some embodiments, the correlation
can be temporal; e.g., the time of emission of the light indicates
the identity of the at least one base, such as is the case when
different base analogs are provided for use in a polymerization
reaction at different times. In some embodiments, the correlation
can be spectral; e.g., the spectrum of the emitted light indicates
the identity of the at least one base, such as is the case when
different base analogs that comprise different fluorophores are
provided for use in a polymerization reaction.
[0131] In some embodiments, single molecule nucleic acid sequencing
comprises multiple sequencing cycles. A sequencing cycle is
understood to mean the events that lead to an emission of light
correlated to the identity of at least one base that would be
repeated in order to identify at least a second base in the nucleic
acid after a first base has been identified. Thus, in methods
according to the present disclosure that comprise single molecule
nucleic acid sequencing, the single molecule nucleic acid
sequencing can comprise at least a given number of sequencing
cycles that lead to at least the given number of emissions of light
correlated collectively to the identity of at least the given
number of bases in the nucleic acid, and the method comprises
identifying at least the given number of bases in the nucleic acid.
In some embodiments, the given number may be, for example, 2, 3, 4,
5, 10, 20, 50, 100, 200, or 500.
[0132] Sequencing methods can comprise determining the identity of
one or more bases in a nucleic acid. In some embodiments of methods
according to the present disclosure, in which performing single
molecule nucleic acid sequencing leads to emission of light that is
detected with at least one light detector comprising at least a
first optical sensor and a second optical sensor, and output signal
from the at least two optical sensors is processed, the identity of
at least one base in a nucleic acid can be determined by comparing
at least one result of the processing with at least one known
result corresponding to at least one known type.
[0133] For example, a result of the processing can indicate a time
at which a reaction occurred; when light emitted is temporally
correlated to the identity of at least one base in the nucleic
acid, said time can be used to identify at least one base in the
nucleic acid.
[0134] In another example, a result of the processing can be a
determination of which fluorophore was incorporated into the
product of a reaction; when light emitted is spectrally correlated
to the identity of at least one base in the nucleic acid, said
determination can be used to identify at least one base in the
nucleic acid.
2.2.2.1 Base Extension Sequencing: Stepwise Extension
[0135] In some embodiments, a detection apparatus provided by the
present disclosure may be used to detect light generated during
base extension sequencing. In some embodiments, base extension
sequencing begins by providing a partial duplex sample nucleic acid
comprising a single stranded nucleic acid to be sequenced, an end
link primer associated with the 3' end of nucleic acid to be
sequenced, and a sequencing primer annealed thereto. In some
embodiments, polymerase and modified nucleotides may be then
applied to the light detection apparatus in a suitable buffer. In
some embodiments, the nucleotides may include a covalently-linked
detectable label, e.g., a fluorescent label, and a blocking group
to prevent any secondary extension. Accordingly, the sequencing
pauses after the addition of a single nucleotide to the 3' end of
sequencing primer.
[0136] In a first step of one embodiment of a base extension
sequencing reaction, a nucleotide with a fluorescent blocking group
may be added by a DNA polymerase to the 3' end of sequencing
primer. In some embodiments, the fluorescent label may act as the
blocking group. In other embodiments, they may be separate
moieties. A single nucleotide may be incorporated at the 3' end of
sequencing primer and is identified by its label by the
corresponding light detector. The fluorescent label and blocking
group are then removed, e.g., by chemical or enzymatic lysis, to
permit additional cycles of base extension. In certain embodiments,
the label and blocking groups may be removed simultaneously or
sequentially and in any order. By compiling the order of the bases
added, the sequence of the sample nucleic acid may be deduced in
the 3' to 5' direction, one base at a time.
[0137] Generally, there are two ways to recognize the nucleotide
added during stepwise extension. In the first case, the four
nucleotides may all have the same detectable label, but are added
one at a time, in a predetermined order. The identity of the
extended nucleotide may be determined by the order that the
nucleotide is added in the extension reaction. In the second mode
for recognizing the base integrated during extension, four
different nucleotides may be added at the same time and each is
coupled with a distinct detectable label. In different embodiments,
the excitation or emission spectra and/or intensity of the labels
may differ. The identity of the nucleotide added in the extension
may be determined by the intensity and/or wavelength (i.e.,
excitation or emission spectra) of the detected label.
2.2.2.2 Sequencing by Synthesis: Multi-Step Extension
[0138] In some embodiments, sequencing by synthesis may proceed
with multiple uninterrupted extensions, e.g., without the use of
blocking groups. In these embodiments, the polymerization reaction
may be monitored by detecting the release of the pyrophosphate
after nucleoside triphosphate hydrolysis, i.e., the release of the
.beta. and y phosphate complex. This complex may be detected
directly, for example, by a fluorescent moiety on the complex, or
indirectly, for example, by coupling the pyrophosphate to a chemi-
or bio-luminescent detection system, as discussed above.
[0139] In some embodiments, the sample nucleic acid may be
sequenced essentially continuously by using
terminal-phosphate-labeled nucleotides. Exemplary embodiments of
terminal-phosphate-labeled nucleotides and methods of their use are
described in, e.g., U.S. Pat. No. 7,361,466 and U.S. Patent
Publication No. 2007/0141598, published Jun. 21, 2007. Briefly, the
nucleotides may be applied to the system provided by the present
disclosure and, when hydrolyzed during the polymerization, the
labeled pyrophosphate may be detected by a corresponding light
detector. In some embodiments, all four nucleotides may comprise
distinct labels and be added simultaneously. In some embodiments,
the nucleotides may comprise indistinguishable, e.g., identical,
labels and be added sequentially in a predetermined order.
Sequential, cyclical addition of nucleotides with indistinguishable
labels still permits multiple, uninterrupted polymerization steps,
e.g., in homopolymer sequences.
2.2.3 Additional Applications
[0140] A detection apparatus consistent with the present disclosure
may simultaneously detect millions of nucleic acid segments. If
each segment is, for example, 1000 bases long, a single device
could obtain upwards of billions of base sequences at once.
Discussed below are additional applications of the apparatuses and
methods provided herein.
2.2.3.1 Whole Genome Sequencing
[0141] A detection apparatus consistent with the present disclosure
may be used to perform whole or partial genome sequencing of, e.g.,
a virus, bacterium, fungi, eukaryote, or vertebrate, e.g., a
mammal, e.g., a human.
[0142] Genomic DNA may be sheared into fragments of at least 20,
50, 100, 200, 300, 500, 800, 1200, 1500 nucleotides, or longer, for
sequencing. In some embodiments, the sheared genomic DNA may be
from 20 to 50, from 20 to 100, from 20 to 500, from 20 to 1000,
from 500 to 1200, or from 500 to 1500 nucleotides long. In some
embodiments, the nucleic acids to be sequenced, along with
associated end link primers, may be made single stranded, annealed
to a sequencing primer, and applied to a system provided by the
present disclosure for sequencing as described above.
2.2.3.2 Gene Expression Profiling
[0143] In other embodiments, a detection apparatus consistent with
the present disclosure may be used to sequence cDNA for gene
expression profiling. For example, mRNA levels may be quantified by
measuring the relative frequency that a particular sequence is
detected on a device. Several million cDNA molecules may be
sequenced in parallel on a device provided by the present
disclosure. If a cell contains, on average, 350,000 mRNA molecules,
a transcript present at even one copy per cell is expected to be
sequenced approximately three times in one million sequencing
reactions. Accordingly, the devices provided by the present
disclosure are suitable for single molecule sequencing with single
copy number sensitivity.
[0144] cDNA synthesis is well known in the art and typically
includes total RNA extraction with optional enrichment of mRNA.
cDNA is produced from mRNA by steps including, for example: reverse
transcription, for first strand synthesis; RNAse treatment, to
remove residual RNA; random hexamer priming of the first strand,
and second strand synthesis by DNA polymerase. The resultant cDNA
is suitable for sequencing on the devices provided by the present
disclosure. Methods of isolating and preparing both DNA and RNA are
well known in the art. See, for example, Joseph Sambrook and David
Russell, Molecular Cloning: A Laboratory Manual Cold Spring Harbor
Laboratory Press; 3rd edition (2001).
2.2.3.3 Additional Detection Methods
[0145] (a) FRET
[0146] In some embodiments, a molecule may be detected on a
detection apparatus provided by the present disclosure by Forster
resonance energy transfer (FRET), sometimes known as fluorescence
resonance energy transfer. As is known in the art, FRET occurs when
an excited donor molecule non-radiatively transfers energy to an
acceptor molecule, which emits the energy, typically as light. FRET
can help reduce background light by, e.g., providing greater
spectral separation between effective excitation and emission
wavelengths for a molecule being detected. FRET is often used to
detect close molecular interactions since its efficiency decays as
the sixth power of the distance between donor and acceptor
molecules. For example, Zhang et al. (Nature Materials 4:826-31
(2005)) detected nucleic acid hybridization by FRET. There, a
biotinylated nucleic acid target was conjugated to an avidin-coated
quantum dot donor, which then excited a Cy5-conjugated DNA probe.
In some embodiments, a labeled capture molecule and labeled sample
molecule may form a donor/acceptor (or vice versa) pair for
detection by FRET.
[0147] In some embodiments of nucleic acid sequencing provided by
the present disclosure, fluorescently labeled nucleotides may act
as acceptor chromophores for a donor chromophore attached to a
polymerase or ligase. Accordingly, in these embodiments, the donor
chromophore located on the polymerase or ligase may excite an
acceptor chromophore on a nucleotide being polymerized on, or
ligated to, the sample nucleic acid. Nucleotides not proximate to
the polymerase may be not excited due to the rapid falloff in FRET
efficiency. In some embodiments the donor molecule may be, another
fluorophore, e.g., a quantum dot. Quantum dots, e.g., semiconductor
quantum dots are known in the art and are described in, e.g.,
International Publication No. WO 03/003015. Means of coupling
quantum dots to, e.g., biomolecules are known in the art, as
reviewed in, e.g., Mednitz et al., Nature Materials 4:235-46 (2005)
and U.S. Patent Publication Nos. 2006/0068506 and 2008/0087843,
published Mar. 30, 2006 and Apr. 17, 2008, respectively. In some
embodiments, quantum dots may be conjugated to a DNA polymerase
molecule. As already discussed above for conjugating enzymes to
linker sites, the skilled artisan will undoubtedly appreciate that
when conjugating fluorophores to, e.g., a DNA polymerase or ligase,
care must be taken to retain enzyme function by mitigating any
effect of conjugating the fluorophore on the primary, secondary,
and tertiary structures of the enzyme.
[0148] (b) Multi Photon Excitation
[0149] In some embodiments, a chromophore may be excited by two or
more photons. For example, in some embodiments, excitation of
either a donor or acceptor chromophore in FRET may be via two or
more photons. Two photon and multi-photon excitation are described
further in, e.g., U.S. Pat. Nos. 6,344,653 and 5,034,613.
[0150] (c) Time Resolved Detection
[0151] In some embodiments, the excitation light source and light
detectors of an apparatus provided by the present disclosure may be
modulated to have a characteristic phase shift. Using methods known
in the art, for example, as disclosed in U.S. Patent Publication
No. 2008/0037008, published Feb. 14, 2008, light emitted from a
molecule being detected on an apparatus provided by the present
disclosure may be measured by a corresponding light detector
without interference from an excitation light source.
[0152] (d) Other Fluorescent Detection Apparatuses and Methods
[0153] In some embodiments, methods of the present disclosure
relate to detection of light emitted by at least one object in a
biological cell, which can be a living or fixed cell. In some
embodiments, the at least one object is chosen from at least one
object comprising at least one quantum dot, at least one object
comprising at least one fluorescent protein, and at least one
object comprising at least one fluorescent small chemical moiety.
In some embodiments, the at least one object is fluorescently
labeled and comprises at least one oligonucleotide, polynucleotide,
oligopeptide, polypeptide, oligosaccharide, polysaccharide, or
lipid.
[0154] In some embodiments, the at least one object comprises a
fixed and limited number of fluorophores, such as at most 20, 10,
5, or 2 fluorophores, which can be chosen from quantum dots,
fluorescent proteins, and fluorescent small chemical moieties. In
some embodiments, the at least one object comprises a single
fluorophore chosen from a quantum dot, a fluorescent protein, and a
fluorescent small chemical moiety. Many examples of fluorescent
small chemical moieties were discussed above. In some embodiments,
fluorescent small chemical moieties may have an emission peak
between 300 and 800 nm and/or a quantum yield (fraction of photons
emitted per photon of peak absorption wavelength absorbed) of at
least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.
2.3 Biomolecule Analysis Service
[0155] The present disclosure also provides a method of providing
biomolecule analysis service using a detection apparatus in
accordance with embodiments consistent with the present disclosure.
In some embodiments, the method may include the steps of providing
a sample including a biomolecule to be analyzed from a service
requester to a service provider and the service requester receiving
analytical results from the service provider, wherein the results
may be produced using an apparatus provided by the present
disclosure. In some embodiments, the method may be performed for
remunerative consideration, e.g., fee-for-service or contract
service agreements. In addition, the sample may be shipped directly
between the service requester and the service provider, or mediated
by a vendor. In some embodiments, the service provider or vendor
may be geographically located in a territory outside of the United
States of America, e.g. in another country.
3. EXAMPLES
3.1 Example 1
[0156] FIGS. 16A-16D show the computer-simulation results for
nanowell 121, 122, 123, and 124 shown in FIG. 3, respectively. In
the simulation, a finite-differential-time-domain (FDTD) method was
used to compute the electric field distribution of the excitation
light propagating along the longitudinal direction of the waveguide
and passing by or through the effective excitation zone. The
strength of the electric field may represent the intensity of the
electromagnetic field of the light in the waveguide. In FIGS.
16A-16D, the strength of the electric field is shown in an
arbitrary unit.
[0157] In order to more closely simulate the actual situation, in
this simulation, a particle having a diameter of 100 nm was assumed
to be near the bottom of the nanowell. The refractive index of the
particle was set to be 2.5. The refractive index of the core layer
was set to be 2.25, the refractive indices of the upper and lower
cladding layers were set to be 1.45, the refractive index of the
sample solution filling the nanowells was set to be 1.33. The
thickness of the core layer was set to be 100 nm. In addition, the
diameter of the bottom of the nanowell was set to be 50 nm for all
four types of nanowells 121, 122, 123, and 124, and the angle of
the side wall of the nanowell with respect to the interface between
the core layer and the upper cladding layer was set to be 60
degree. For the simulation for nanowell 121, the bottom of the
nanowell was set to be 50 nm away from the interface between the
cladding layer and the core layer. For the simulation for nanowell
123, the depth of the nanowell extending in the core layer was set
to be 50 nm. That is, the bottom surface of nanowell 123 was set to
be located at the center of the core layer. The figures on the
left-hand-side of each of FIGS. 16A-16D shows the time-averaged
electric field distribution in the waveguide with different
nanowells 121, 122, 123, and 124, respectively. The figures on the
right-hand-side of each of FIGS. 16A-16D shows the electric field
distribution along the vertical direction in the waveguide with
different nanowells 121, 122, 123, and 124, respectively.
3.2 Example 2
[0158] This example illustrates the simulation results of the
transmittances of a TE mode light having a wavelength of 473 nm and
TM mode light having a wavelength of 550 nm through a metal pattern
as in the structure shown in FIG. 5 as functions of grating period
and depth of the metal pattern, respectively.
[0159] FIG. 17A shows the simulated transmittances of the TE mode
light through the metal pattern versus the grating period of the
metal pattern. Different curves in FIG. 17A represent results
computed at different values of metal pattern depth. FIG. 17B shows
the simulated transmittances of the TM mode light through the metal
pattern versus the grating period of the metal pattern. Different
curves in FIG. 17B represent results computed at different values
of metal pattern depth.
[0160] It is seen from FIG. 17B that, when the grating period of
the metal pattern is smaller than 300 nm, the transmittance of the
TM mode light is higher than about 30%, which is also much higher
than the transmittance of the TE mode light. Therefore, it can be
expected that a high SNR (i.e., the ratio between the
transmittances of the TM mode light and the TE mode light) may be
realized with a grating period smaller than 300 nm. FIG. 17C shows
the SNR versus the grating period of the metal pattern computed at
different values of metal pattern depth. It can be seen that an SNR
larger than 10 can be obtained at, for example a grating period of
250 nm and a depth of 150 nm. Furthermore, when the grating period
and the depth are 110 nm and 250 nm, respectively, the SNR is
larger than 10.sup.7.
3.3 Example 3
[0161] This example illustrates calculated transmittances of a TE
mode light and a TM mode light through the metal pattern in the
structure shown in FIG. 5. In this example, nanostructured metal
pattern 152 is made of aluminum and the lower protection layer 144
surrounding the aluminum pattern is made of silicon oxide. The
periodicity and depth of the aluminum pattern are 110 nm and 245
nm, respectively. FIGS. 18A and 18B show the transmittances of the
TE mode light and the TM mode light as a function of incident
angle, respectively. It is seen from FIGS. 18A and 18B that, the
transmittance of the TE mode light decreases from about 10.sup.-8
to about 10.sup.-12 when the absolute value of the incident angle
increases from 0 degree to larger than 60 degree, whereas the
transmittance of the TM mode light is always higher than 60% within
the range of the incident angle from about -60 degree to about 60
degree.
3.4 Example 4
[0162] FIG. 19 shows an FDTD simulation result of a prism coupler
shown in FIG. 20. In this simulation, the gap between the prism and
the waveguide was set to be 73 nm. Polycarbonate (n=1.6) was used
as the material for the core layer, and the thickness of the core
layer was set to be 2 .mu.m. The incident light (A=430 nm) with TM
mode was used. Experiments were also conducted to measure the
coupling efficiency of the prism coupler, which may be, for
example, about 60%. For example, if the light source, such as a
He--Ne laser, emits a light with a power of about 2 mW, the power
of the light coupled into the waveguide as the excitation light may
be about 1.2 mW.
3.5 Example 5
[0163] FIG. 21 shows the simulated speckle of the incident light
coupled into the waveguide 110 shown in FIG. 7. For this
simulation, cylindrical lens made of PMMA with aperture diameter of
0.5 mm and thickness of 0.2 mm was used for laser beam shaping. The
projection distance from the lens to waveguide is 1 mm. See FIGS.
22A and 22B. The -thickness of the speckle was about 300 nm and the
coupling efficiency was about 70%. For example, if the light
source, such as a He--Ne laser, emits a light with a power of about
2 mW, the power of the light coupled into the waveguide as the
excitation light may be about 1.4 mW.
3.6 Example 6
[0164] FIG. 23 shows, as an example, the measured dependence of the
power of the light coupled into the core layer 112 on the thickness
of the lower cladding layer 116 for the waveguide shown in FIG. 9.
In this measurement, the grating period was about 410 nm and the
thickness of the core layer was about 100 nm. The wavelength of the
incident light was about 633 nm. In FIG. 23, the power of the light
coupled into the core layer is normalized with respect to the
incident power.
3.7 Example 7
[0165] An FDTD simulation was performed on the grating couplers
shown in FIGS. 8 and 9, respectively. In the simulation, the
periodicity of the first grating in FIGS. 8 and 9 was set to be 410
nm and the periodicity of the second grating in FIG. 9 was set to
be 300 nm. The depths of both gratings were set to be 55 nm. The
incident light was set to have a wavelength of 473 nm and a beam
radius of 5 .mu.m; the refractive indices of the core and cladding
layers were set to be 2.196 and 1.445, respectively. FIGS. 24 and
25 show the simulated instantaneous electric field distribution in
the structures shown in FIGS. 8 and 9, respectively. The calculated
coupling efficiencies for the structures shown in FIGS. 8 and 9
were 20% and 25%, respectively, which confirms the increasing of
coupling efficiencies by adding the second grating 404.
3.8 Example 8
[0166] A calculation was performed on such a structure shown in
FIG. 11. For the calculation, the effective refractive index of the
grating 406 was set to be 2.2, the refractive indices of the core
layer 112 and the cladding layers 114 and 116 were set to be 1.6
and 1.45, respectively, and the periodicity and depth of the
grating were set to be 321 nm and 120 nm, respectively. Under such
conditions, for an incident light with a wavelength of 473 nm and
normally incident onto the grating 406, the calculated coupling
efficiency was 29%.
3.9 Example 9
[0167] Table 1 shows the calculation results of the power density
of the light emitted from an absorbing-emitting light coupler using
phthalocyanine,
4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran
(DCM) doped polyvinylphyrodione (PVP) as the photoluminescence
material under different conditions. It is seen from Table 1 that,
for a same incident light, the power density of the light coupled
into the waveguide may be as high as 37.67 W/cm.sup.2.
TABLE-US-00001 TABLE 1 DCM doped DCM doped DCM doped
Photoluminescence material PVP PVP PVP d.sub.1 (cm) 0.0002 0.0002
0.0002 d.sub.2 (cm) 2.0 2.0 2.0 L (cm) 3.0 3.0 3.0
Photoluminescence material 0.04 0.04 0.04 concentration (M)
.lamda..sub.1 (nm) 465 465 465 .lamda..sub.2 (nm) 630 630 630
Stokes shift (.lamda..sub.1 - .lamda..sub.2, nm) 165 165 165
P.sub.0(.lamda..sub.1) (W) 1.0 1.0 1.0 Power density of incident
1.0 1.0 1.0 light at .lamda..sub.1 (W/cm.sup.2) .phi..sub.FL 0.6
0.6 0.6 R.sub.1 0.90 0.90 0.90 .eta..sub.c 0.0850 0.0850 0.1850
P(.lamda..sub.2)/P.sub.0(.lamda..sub.1) 0.0060 0.0103 0.0226 Power
density of excitation 9.92 17.17 37.67 light at .lamda..sub.2
(W/cm.sup.2)
3.10 Example 10
[0168] A DNA molecule is sequenced using the detection apparatus
disclosed herein. The detection apparatus of this example comprises
a light source, a light coupler, a planar waveguide having a
nanowell array formed in the upper cladding layer of the waveguide,
and a detector array formed beneath the waveguide.
[0169] In this example, the light source is a He--Ne laser emitting
a light having a wavelength of about 633 nm. The power of the
He--Ne laser is about 2 mW. The light coupler is a side coupler
consisting of a cylindrical lens made of PMMA. The cylindrical lens
has an aperture diameter of about 0.5 mm and a thickness of about
0.2 mm, and is arranged at a distance of about 1 mm from the side
of the waveguide. The detector array consists of 1000 light
detectors, each of which is a silicon photodiode.
[0170] The planar waveguide comprises a core layer having a
thickness of about 100 nm, an upper cladding layer, and a lower
cladding layer. The core layer is made of silicon nitride having a
refractive index of about 2.05. The upper and lower cladding layers
are made of silicon oxide having a refractive index of about 1.46.
The nanowell array formed in the upper cladding layer contains 1000
nanowells. For each of the nanowells, there is a light detector
used to detect the light emitted by the molecule trapped in the
nanowell.
[0171] Each nanowell has a funnel shape with a circular horizontal
cross-section. The nanowells extend through the full thickness of
the upper cladding layer so as to expose the core layer. The
diameter of the bottom of the nanowell is about 50 nm. The angle
between the sidewall of the nanowell and the vertical direction is
about 30 degrees. The effective excitation zone formed in each
nanowell is about 1 atto liter (al).
[0172] Nucleic acid polymerases are chemically attached to the
bottom surfaces of nanowells with an average density of about 1
active, accessible polymerases per nanowell effective excitation
zone.
[0173] A solution of circular, single-stranded DNA molecules with
an average length of 200 nt at a concentration of 0.1 molecules per
atto liter in a suitable sequencing reaction buffer is applied to
the detection apparatus. The circular DNA molecules contain a known
insert sequence of approximately 20 nt 3' to an unknown sample
sequence. A sequencing primer complementary to the known insert
sequence and fluorescently labeled dNTP analogs with blocking
groups suitable for reversible terminator sequencing by synthesis
are provided. In a plurality of nanowells, a ternary complex of a
polymerase, DNA molecule, and sequencing primer is formed and the
polymerase adds one fluorescently labeled dNTP analog to the 3' end
of the sequencing primer.
[0174] Light emitted from the He--Ne laser is partially coupled
into the waveguide by the side coupler. Some of the light coupled
into the waveguide propagates in the core layer, acting as the
excitation light. In the plurality of nanowells, a fluorescently
labeled dNTP analog is excited by the excitation light entering the
effective excitation zones formed near the bottom of the nanowells
and emits fluorescent light. This fluorescent light is detected by
the detectors, which in turn generate output signals to be
processed to identify the base comprised by the nucleotide analog
added to the sequencing primer.
[0175] In the plurality of nanowells, the fluorophore and the
blocking group are chemically removed. The polymerase then adds
another fluorescently labeled dNTP analog, which is detected as
above and then removed. This cycle is repeated a sufficient number
of times to acquire a sequencing read at least twice the length of
the DNA molecule (i.e., the DNA molecule is sequenced and
resequenced).
* * * * *